Oils, lipids and fatty acids produced in transgenic brassica plant

ABSTRACT

The invention relates to a method for producing eicosapentanoic acid, docosapentanoic acid and/or docohexanoic acid in transgenic plants. According to said method, the plant is provided with at least one nucleic acid sequence coding for a polypetide with a Δ6 desaturase activity, at least one nucleic acid sequence coding for a polypeptide with a Δ6 elongase activity, at least one nucleic acid sequence coding for a polypeptide with a Δ5 desaturase activity, and at least one nucleic acid sequence coding for a polypeptide with a Δ5 elongase activity, the nucleic acid sequence coding for a polypeptide with a Δ5 elongase activity being modified in relation to the nucleic acid sequence in the organism from which the sequence originates, such that it is adapted to the codon use in at least one type of plant. For the production of docosahexanoic acid, at least one nucleic acid sequence coding for a polypeptide with a Δ4 desaturase activity is also introduced into the plant.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 16/371,696filed Apr. 1, 2019, which is a continuation of application Ser. No.15/256,914, filed Sep. 6, 2016, now U.S. Pat. No. 10,301,638, which is acontinuation of application Ser. No. 12/280,090, now U.S. Pat. No.10,190,131, which is a national stage application (under 35 U.S.C. §371) of PCT/EP2007/051675, filed Feb. 21, 2007, which claims benefit ofGerman application 10 2006 008 030.0, filed Feb. 21, 2006 and Europeanapplication 06120309.7, filed Sep. 7, 2006. The entire content of eachaforementioned application is hereby incorporated by reference in itsentirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 57672D_Seqlisting. The size of the text file is809,688 bytes, and the text file was created on Oct. 30, 2020.

The present invention relates to a process for the production ofeicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoic acidin transgenic plants, providing in the plant at least one nucleic acidsequence which codes for a polypeptide having a Δ6-desaturase activity;at least one nucleic acid sequence which codes for a polypeptide havinga Δ6-elongase activity; at least one nucleic acid sequence which codesfor a polypeptide having a Δ5-desaturase activity; and at least onenucleic acid sequence which codes for a polypeptide having a Δ5-elongaseactivity, where the nucleic acid sequence which codes for a polypeptidehaving a Δ5-elongase activity is modified by comparison with the nucleicacid sequence in the organism from which the sequence is derived in thatit is adapted to the codon usage in one or more plant species.

In a preferred embodiment there is additionally provision of furthernucleic acid sequences which code for a polypeptide having the activityof an ω3-desaturase and/or of a Δ4-desaturase in the plant.

In a further preferred embodiment there is provision of further nucleicacid sequences which code for acyl-CoA dehydrogenase(s), acyl-ACP (acylcarrier protein) desaturase(s), acyl-ACP thioesterase(s), fatty acidacyl transferase(s), acyl-CoA: lysophospholipid acyl transferase(s),fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, alleneoxide synthases, hydroperoxide lyases or fatty acid elongase(s) in theplant.

The invention furthermore relates to recombinant nucleic acid moleculescomprising at least one nucleic acid sequence which codes for apolypeptide having a Δ6-desaturase activity; at least one nucleic acidsequence which codes for a polypeptide having a Δ5-desaturase activity;at least one nucleic acid sequence which codes for a polypeptide havinga Δ6-elongase activity; and at least one nucleic acid sequence whichcodes for a polypeptide having a Δ5-elongase activity and which ismodified by comparison with the nucleic acid sequence in the organismfrom which the sequence originates in that it is adapted to the codonusage in one or more plant species.

A further part of the invention relates to oils, lipids and/or fattyacids which have been produced by the process according to theinvention, and to their use.

Finally, the invention also relates to transgenic plants which have beenproduced by the process of the invention or which comprise a recombinantnucleic acid molecule of the invention, and to the use thereof asfoodstuffs or feedstuffs.

Lipid synthesis can be divided into two sections: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Usual lipids which are used inmembranes comprise phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACPby acetyl transacylase. After condensation reaction, these two productmolecules together form acetoacetyl-ACP, which is converted via a seriesof condensation, reduction and dehydration reactions so that a saturatedfatty acid molecule with the desired chain length is obtained. Theproduction of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (regarding the fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., p. 612-636 and references citedtherein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme:Stuttgart, N.Y., and the references therein, and Magnuson, K., et al.(1993) Microbiological Reviews 57:522-542 and the references therein).To undergo the further elongation steps, the resultingphospholipid-bound fatty acids must be returned to the fatty acid CoAester pool. This is made possibly by acyl-CoA:lysophospholipidacyltransferases. Moreover, these enzymes are capable of transferringthe elongated fatty acids from the CoA esters back to the phospholipids.If appropriate, this reaction sequence can be followed repeatedly.

Furthermore, fatty acids must subsequently be transported to variousmodification sites and incorporated into the triacylglycerol storagelipid. A further important step during lipid synthesis is the transferof fatty acids to the polar head groups, for example by glycerol fattyacid acyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).

An overview of the biosynthesis of fatty acids in plants, desaturation,the lipid metabolism and the membrane transport of lipidic compounds,beta-oxidation, the modification of fatty acids, cofactors and thestorage and assembly of triacylglycerol, including the references isgiven by the following papers: Kinney (1997) Genetic Engineering, Ed.: JK Setlow, 19:149-166; Ohlrogge and Browse (1995) Plant Cell 7:957-970;Shanklin and Cahoon (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol.49:611-641; Voelker (1996) Genetic Engeneering, Ed.: J K Setlow,18:111-13; Gerhardt (1992) Prog. Lipid R. 31:397-417; Gühnemann-Schäfer& Kindl (1995) Biochim. Biophys Acta 1256:181-186; Kunau et al. (1995)Prog. Lipid Res. 34:267-342; Stymne et al. (1993) in: Biochemistry andMolecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murataand Somerville, Rockville, American Society of Plant Physiologists,150-158; Murphy & Ross (1998) Plant Journal. 13(1):1-16.

Depending on the desaturation pattern, two large classes ofpolyunsaturated fatty acids, the ω6 and the ω3 fatty acids, which differwith regard to their metabolism and their function, can bedistinguished.

In the text which follows, polyunsaturated fatty acids are referred toas PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA,long chain poly unsaturated fatty acids, LCPUFA).

The fatty acid linoleic acid (18:2^(Δ9,12)) acts as starting materialfor the ω6 metabolic pathway, while the ω3 pathway proceeds vialinolenic acid (18:3^(Δ9,12,15)). Linolenic acid is formed from linoleicacid by the activity of an ω3-desaturase (Tocher et al. (1998) Prog.Lipid Res. 37: 73-117; Domergue et al. (2002) Eur. J. Biochem. 269:4105-4113).

Mammals, and thus also humans, have no corresponding desaturase activity(Δ12- and ω3-desaturase) for the formation of the starting materials andmust therefore take up these fatty acids (essential fatty acids) via thefood. Starting with these precursors, the physiologically importantpolyunsaturated fatty acids arachidonic acid (=ARA, 20:4^(Δ5,8,11,14))an ω6-fatty acid and the two ω3-fatty acids eicosapentaenoic acid (=EPA,20:5^(Δ5,8,11,14,17)) and docosahexaenoic acid (DHA,22:6^(Δ4,7,10,13,17,19)) are synthesized via a sequence of desaturaseand elongase reactions.

The elongation of fatty acids, by elongases, by 2 or 4 C atoms is ofcrucial importance for the production of C₂₀- and C₂₂-PUFAs,respectively. This process proceeds via 4 steps. The first step is thecondensation of malonyl-CoA onto the fatty acid acyl-CoA by ketoacyl-CoAsynthase (KCS, hereinbelow referred to as elongase). This is followed bya reduction step (ketoacyl-CoA reductase, KCR), a dehydratation step(dehydratase) and a final reduction step (enoyl-CoA reductase). It hasbeen postulated that the elongase activity affects the specificity andrate of the entire process (Millar and Kunst (1997) Plant Journal12:121-131).

Fatty acids and triacylglycerides have a multiplicity of applications inthe food industry, in animal nutrition, in cosmetics and thepharmacological sector. Depending on whether they are free saturated orunsaturated fatty acids or else triacylglycerides with an elevatedcontent of saturated or unsaturated fatty acids, they are suitable forvery different applications. Thus, for example, lipids with unsaturated,specifically with polyunsaturated fatty acids, are preferred in humannutrition. The polyunsaturated ω3-fatty acids are supposed to have apositive effect on the cholesterol level in the blood and thus on theprevention of heart disease. The risk of heart disease, strokes orhypertension can be reduced markedly by adding these ω3-fatty acids tothe food (Shimikawa (2001) World Rev. Nutr. Diet. 88: 100-108).

ω3-fatty acids also have a positive effect on inflammatory, specificallyon chronically inflammatory, processes in association with immunologicaldiseases such as rheumatoid arthritis (Calder (2002) Proc. Nutr. Soc.61: 345-358; Cleland and James (2000) J. Rheumatol. 27: 2305-2307). Theyare therefore added to foodstuffs, specifically to dietetic foodstuffs,or are employed in medicaments. ω6-fatty acids such as arachidonic acidtend to have a negative effect in connection with these rheumatologicaldiseases.

ω3- and ω6-fatty acids are precursors of tissue hormones, known aseicosanoids, such as the prostaglandins, which are derived fromdihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, andof the thromboxanes and leukotrienes, which are derived from arachidonicacid and eicosapentaenoic acid. Eicosanoids (known as the PG₂ series)which are formed from the ω6-fatty acids, generally promote inflammatoryreactions, while eicosanoids (known as the PG₃ series) from ω3-fattyacids have little or no proinflammatory effect.

Polyunsaturated long-chain ω3-fatty acids such as eicosapentaenoic acid(=EPA, C20:5^(Δ5,8,11,14,17)) or docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) are important components of human nutritionowing to their various roles in health aspects, including thedevelopment of the child brain, the functionality of the eyes, thesynthesis of hormones and other signal substances, and the prevention ofcardiovascular disorders, cancer and diabetes (Poulos, A (1995) Lipids30:1-14; Horrocks, L A and Yeo Y K (1999) Pharmacol Res 40:211-225).

Owing to the present-day composition of human food, an addition ofpolyunsaturated ω3-fatty acids, which are preferentially found in fishoils, to the food is particularly important. Thus, for example,polyunsaturated fatty acids such as docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) or eicosapentaenoic acid (=EPA,C20:5^(Δ5,8,11,14,17)) are added to infant formula to improve thenutritional value. There is therefore a demand for the production ofpolyunsaturated long-chain fatty acids.

The various fatty acids and triglycerides are mainly obtained frommicroorganisms such as Mortierella or Schizochytrium or fromoil-producing plants such as soybeans, oilseed rape, and algae such asCrypthecodinium or Phaeodactylum and others, being obtained, as a rule,in the form of their triacylglycerides (=triglycerides=triglycerols).However, they can also be obtained from animals, for example, fish. Thefree fatty acids are advantageously prepared by hydrolyzing thetriacylglycerides. Very long-chain polyunsaturated fatty acids such asDHA, EPA, arachidonic acid (ARA, C20:4^(Δ5,8,11,14)) dihomo-γ-linolenicacid (DHGL, C20:3^(Δ8,11,14)) or docosapentaenoic acid (DPA,C22:5^(Δ7,10,13,16,19)) are, however, not synthesized in oil crops suchas oilseed rape, soybeans, sunflowers and safflower. Conventionalnatural sources of these fatty acids are fish such as herring, salmon,sardine, redfish, eel, carp, trout, halibut, mackerel, zander or tuna,or algae.

Owing to the positive characteristics of the polyunsaturated fattyacids, there has been no lack of attempts in the past to make availablegenes which are involved in the synthesis of these fatty acids ortriglycerides for the production of oils in various organisms with amodified content of unsaturated fatty acids. Thus, WO 91/13972 and itsUS equivalent describe a Δ9-desaturase. WO 93/11245 claims aΔ15-desaturase and WO 94/11516 a Δ12-desaturase. Further desaturates aredescribed, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al. (1990) J. Biol.Chem., 265: 20144-20149, Wada et al. (1990) Nature 347: 200-203 or Huanget al. (1999) Lipids 34: 649-659. However, the biochemicalcharacterization of the various desaturases has been insufficient todate since the enzymes, being membrane-bound proteins, present greatdifficulty in their isolation and characterization (McKeon et al. (1981)Methods in Enzymol. 71: 12141-12147, Wang et al. (1988) Plant Physiol.Biochem., 26: 777-792).

As a rule, membrane-bound desaturases are characterized by beingintroduced into a suitable organism which is subsequently analyzed forenzyme activity by analyzing the starting materials and the products.Δ6-Desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, WO96/21022, WO 00/21557 and WO 99/27111. The application of this enzymefor the production of fatty acids in transgenic organisms is describedin WO 98/46763, WO 98/46764 and WO 98/46765. The expression of variousdesaturases and the formation of polyunsaturated fatty acids is alsodescribed and claimed in WO 99/64616 or WO 98/46776. As regards theexpression efficacy of desaturases and its effect on the formation ofpolyunsaturated fatty acids, it must be noted that the expression of asingle desaturase as described to date has only resulted in low contentsof unsaturated fatty acids/lipids such as, for example, γ-linolenic acidand stearidonic acid.

There have been a number of attempts in the past to obtain elongasegenes. Millar and Kunst (1997) Plant Journal 12:121-131 and Millar etal. (1999) Plant Cell 11:825-838 describe the characterization of plantelongases for the synthesis of monounsaturated long-chain fatty acids(C22:1) and for the synthesis of very long-chain fatty acids for theformation of waxes in plants (C₂₈-C₃₂). The synthesis of arachidonicacid and EPA is described, for example, in WO 01/59128, WO 00/12720, WO02/077213 and WO 02/08401. The synthesis of polyunsaturated C24-fattyacids is described, for example, in Tvrdik et al. (2000) J. Cell Biol.149:707-718 or in WO 02/44320.

Especially suitable microorganisms for the production of PUFAs aremicroalgae such as Phaeodactylum tricornutum, Porphiridium species,Thraustochytrium species, Schizochytrium species or Crypthecodiniumspecies, ciliates such as Stylonychia or Colpidium, fungi such asMortierella, Entomophthora or Mucor and/or mosses such asPhyscomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen(1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22:1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology73: 269-278). Strain selection has resulted in the development of anumber of mutant strains of the microorganisms in question which producea series of desirable compounds including PUFAs. However, the mutationand selection of strains with an improved production of a particularmolecule such as the polyunsaturated fatty acids is a time-consuming anddifficult process. Moreover, only limited amounts of the desiredpolyunsaturated fatty acids such as DPA, EPA or ARA can be produced withthe aid of the abovementioned microorganisms; in addition, they aregenerally obtained as fatty acid mixtures. This is why recombinantmethods are preferred whenever possible.

Higher plants comprise polyunsaturated fatty acids such as linoleic acid(C18:2) and linolenic acid (C18:3). ARA, EPA and DHA are found not atall in the seed oil of higher plants, or only in miniscule amounts (E.Ucciani: Nouveau Dictionnaire des Huiles Vegetales [New Dictionary ofthe Vegetable Oils]. Technique & Documentation—Lavoisier, 1995. ISBN:2-7430-0009-0). However, the production of LCPUFAs in higher plants,preferably in oil crops such as oilseed rape, linseed, sunflowers andsoybeans, would be advantageous since large amounts of high-qualityLCPUFAs for the food industry, animal nutrition and pharmaceuticalpurposes might be obtained economically. To this end, it is advantageousto introduce, into oilseeds, genes which encode enzymes of the LCPUFAbiosynthesis via recombinant methods and to express them therein. Thesegenes encode for example Δ6-desaturases, Δ6-elongases, Δ5-desaturases orΔ4-desaturases. These genes can advantageously be isolated frommicroorganisms and lower plants which produce LCPUFAs and incorporatethem in the membranes or triacylglycerides. Thus, it has already beenpossible to isolate Δ6-desaturase genes from the moss Physcomitrellapatens and Δ6-elongase genes from P. patens and from the nematode C.elegans.

Transgenic plants which comprise and express genes encoding LCPUFAbiosynthesis enzymes and which, as a consequence, produce LCPUFAs havebeen described, for example, in DE-A-102 19 203 (process for theproduction of polyunsaturated fatty acids in plants). However, theseplants produce LCPUFAs in amounts which require further optimization forprocessing the oils which are present in the plants. Thus, the ARAcontent in the plants described in DE-A-102 19 203 is only 0.4 to 2% andthe EPA content only 0.5 to 1%, in each case based on the total lipidcontent of the plant.

To make possible the fortification of food and of feed withpolyunsaturated, long-chain fatty acids, there is therefore a great needfor a simple, inexpensive process for the production of polyunsaturated,long-chain fatty acids, specifically in plant systems.

One object of the invention is therefore to provide a process with whichlong-chain polyunsaturated fatty acids, especially eicosapentaenoicacid, docosapentaenoic acid and/or docosahexaenoic acid can be producedin large quantities and inexpensively in transgenic plants.

It has now surprisingly been found that the yield of long-chainpolyunsaturated fatty acids, especially eicosapentaenoic,docosapentaenoic acid and/or docosahexaenoic acid, can be increased byexpressing an optimized Δ5-elongase sequence in transgenic plants.

The PUFAs produced by the process of the invention comprise a group ofmolecules which higher animals are no longer able to synthesize and thusmust consume, or which higher animals are no longer able to producethemselves in sufficient amounts and thus must consume additionalamounts thereof, although they can easily be synthesized by otherorganisms such as bacteria.

Accordingly, the object of the invention is achieved by the process ofthe invention for producing eicosapentaenoic acid, docosapentaenoic acidand/or docosahexaenoic acid in a transgenic plant, comprising theprovision in the plant of at least one nucleic acid sequence which codesfor a polypeptide having a Δ6-desaturase activity; at least one nucleicacid sequence which codes for a polypeptide having a Δ6-elongaseactivity; at least one nucleic acid sequence which codes for apolypeptide having a Δ5-desaturase activity; and at least one nucleicacid sequence which codes for a polypeptide having a Δ5-elongaseactivity, where the nucleic acid sequence which codes for a polypeptidehaving a Δ5-elongase activity is modified by comparison with the nucleicacid sequence in the organism from which the sequence is derived in thatit is adapted to the codon usage in one or more plant species. Toproduce DHA it is additionally necessary to provide at least one nucleicacid sequence which codes for a polypeptide having a Δ4-desaturaseactivity in the plant.

The “provision in the plant” means in the context of the presentinvention that measures are taken so that the nucleic acid sequencescoding for a polypeptide having a Δ6-desaturase activity, a polypeptidehaving a Δ6-elongase activity, a polypeptide having a Δ5-desaturaseactivity and a polypeptide having a Δ5-elongase activity are presenttogether in one plant. The “provision in the plant” thus comprises theintroduction of the nucleic acid sequences into the plant both bytransformation of a plant with one or more recombinant nucleic acidmolecules which comprise said nucleic acid sequences, and by crossingsuitable parent plants which comprise one or more of said nucleic acidsequences.

The nucleic acid sequence which codes for a polypeptide having aΔ5-elongase activity is modified according to the invention bycomparison with the nucleic acid sequence in the organism from which thesequence originates in that it is adapted to the codon usage in one ormore plant species. This means that the nucleic acid sequence has beenspecifically optimized for the purpose of the invention without theamino acid sequence encoded by the nucleic acid sequence having beenaltered thereby.

The genetic code is redundant because it uses 61 codons in order tospecify 20 amino acids. Therefore, most of the 20 proteinogenic aminoacids are therefore encoded by a plurality of triplets (codons). Thesynonymous codons which specify an individual amino acid are, however,not used with the same frequency in a particular organism; on thecontrary there are preferred codons which are frequently used, andcodons which are used more rarely. These differences in codon usage areattributed to selective evolutionary pressures and especially theefficiency of translation. One reason for the lower translationefficiency of rarely occurring codons might be that the correspondingaminoacyl-tRNA pools are exhausted and thus no longer available forprotein synthesis.

In addition, different organisms prefer different codons. For thisreason, for example, the expression of a recombinant DNA derived from amammalian cell frequently proceeds only suboptimally in Escherichia coli(E. coli) cells. It is therefore possible in some cases to increaseexpression by replacing rarely used codons with frequently used codons.Without wishing to be bound to one theory, it is assumed that thecodon-optimized DNA sequences make more efficient translation possible,and the mRNAs formed therefrom possibly have a greater half-life in thecell and therefore are available more frequently for translation. Fromwhat has been said above, it follows that codon optimization isnecessary only if the organism in which the nucleic acid sequence is tobe expressed differs from the organism from which the nucleic acidsequence is originally derived.

For many organisms of which the DNA sequence of a relatively largenumber of genes is known there are tables from which the frequency ofuse of particular codons in the respective organism can be taken. It ispossible with the aid of these tables to translate protein sequenceswith relatively high accuracy back into a DNA sequence which comprisesthe codons preferred in the respective organism for the various aminoacids of the protein. Tables on codon usage can be found inter alia atthe following Internet address: kazusa.or.ip/Kodon/E.html. In addition,several companies provide software for gene optimization, such as, forexample, Entelechon (Software Leto) or Geneart (Software GeneOptimizer).

Adaptation of the sequences to the codon usage in a particular organismcan take place with the aid of various criteria. On the one hand, it ispossible to use for a particular amino acid always the codon whichoccurs most frequently in the selected organism but, on the other hand,the natural frequency of the various codons can also be taken intoaccount, so that all the codons for a particular amino acid areincorporated into the optimized sequence according to their naturalfrequency. Selection of the position at which a particular base tripletis used can take place at random in this case. The DNA sequence wasadapted according to the invention taking account of the naturalfrequency of individual codons, it also being suitable to use the codonsoccurring most frequently in the selected organism.

It is particularly preferred for a nucleic acid sequence fromOstreococcus tauri which codes for a polypeptide having a Δ5-elongaseactivity, such as, for example, the polypeptide depicted in SEQ ID NO:110, to be adapted at least to the codon usage in oilseed rape, soybeanand/or flax. The nucleic acid sequence originally derived fromOstreococcus tauri is preferably the sequence depicted in SEQ ID NO:109. The DNA sequence coding for the Δ5-elongase is adapted in at least20% of the positions, preferably in at least 30% of the positions,particularly preferably in at least 40% of the positions and mostpreferably in at least 50% of the positions to the codon usage inoilseed rape, soybean and/or flax.

The nucleic acid sequence used is most preferably the sequence indicatedin SEQ ID NO: 64.

It will be appreciated that the invention also encompasses thosecodon-optimized DNA sequences which code for a polypeptide having theactivity of a Δ5-elongase and whose amino acid sequence is modified inone or more positions by comparison with the wild-type sequence butwhich still has substantially the same activity as the wild-typeprotein.

The nucleic acid sequence which codes for a polypeptide having aΔ6-desaturase activity is preferably selected from the group consistingof:

a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39or 41, preferably having the sequence depicted in SEQ ID NO: 1,b)nucleic acid sequences which code for the amino acid sequence indicatedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40 or 42, preferably in SEQ ID SEQ ID NO: 2,

c) nucleic acid sequences which hybridize with the complementary strandof the nucleic acid sequences indicated a) or b) above, in particular ofthe nucleic acid sequence indicated in SEQ ID NO: 1, under stringentconditions,

d) nucleic acid sequences which are at least 60%, 65%, 70%, 75% or 80%,preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%,particularly preferably at least 91%, 92%, 93%, 94% or 95% andespecially at least 96%, 97%, 98% or 99%, identical to the nucleic acidsequences indicated in a) or b) above, especially to the sequenceindicated in SEQ ID NO: 1, and

e) nucleic acid sequences which code for an amino acid sequence andwhich have at least one, for example 2, 3, 4, 5, 6, 7 or 8, preferablyall of the amino acid pattern indicated in SEQ ID NO: 43, 44, 45, 46,47, 48, 49 or 50.

Amino acid pattern means short amino acid sequences which preferablycomprise less than 50, particularly preferably less than 40 andespecially from 10 to 40 and even more preferably from 10 to 30 aminoacids.

For the present invention, the identity is ascertained preferably overthe full length of the nucleotide or amino acid sequences of theinvention, for example for the nucleic acid sequence indicated in SEQ IDNO: 64 over the full length of 903 nucleotides.

The nucleic acid sequence which codes for a polypeptide having aΔ6-elongase activity is preferably selected from the group consistingof:

a) nucleic acid sequences having the sequence depicted in SEQ ID NO:171, 173, 175, 177, 179, 181 or 183, especially having the sequencedepicted in SEQ ID NO: 171,

b) nucleic acid sequences which code for the amino acid sequenceindicated in SEQ ID NO: 172, 174, 176, 178, 180, 182 or 184, especiallyfor the amino acid sequence indicated in SEQ ID NO: 172,

c) nucleic acid sequences which hybridize with the complementary strandof the nucleic acid sequences indicated a) or b) above, especially ofthe nucleic acid sequence indicated in SEQ ID NO: 1, under stringentconditions,

d) nucleic acid sequences which are at least 60%, 65%, 70%, 75% or 80%,preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%,particularly preferably at least 91%, 92%, 93%, 94% or 95% andespecially at least 96%, 97%, 98% or 99%, identical to the nucleic acidsequences indicated in a) or b) above, especially to the sequenceindicated in SEQ ID NO: 171, and

e) nucleic acid sequences which code for an amino acid sequence andwhich have at least one, for example 2, 3, 4, 5, 6, 7 or 8, preferablyall of the amino acid pattern indicated in SEQ ID NO: 185, 186, 187,188, 189, 190, 191 or 192.

The nucleic acid sequence which codes for a polypeptide having aΔ6-elongase activity is in particular likewise a codon-optimizedsequence according to the present invention, preferably the nucleic acidsequence depicted in SEQ ID NO: 122.

The nucleic acid sequence which codes for a polypeptide having aΔ5-desaturase activity is preferably selected from the group consistingof:

a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 51,53 or 55, preferably having the sequence depicted in SEQ ID NO: 51,

b) nucleic acid sequences which code for the amino acid sequenceindicated in SEQ ID NO: 52, 54 or 56, preferably for the amino acidsequence indicated in SEQ ID NO: 52,

c) nucleic acid sequences which hybridize with the complementary strandof the nucleic acid sequences indicated in a) or b) above, especially ofthe nucleic acid sequence indicated in SEQ ID NO: 51, under stringentconditions,

d) nucleic acid sequences which are at least 60%, 65%, 70%, 75% or 80%,preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%,particularly preferably at least 91%, 92%, 93%, 94% or 95% andespecially at least 96%, 97%, 98% or 99%, identical to the nucleic acidsequences indicated in a) or b) above, especially to the nucleic acidindicated under SEQ ID NO: 51, and

e) nucleic acid sequences which code for an amino acid sequence whichhave at least one, for example 2, 3, 4, 5, 6 or 7, preferably all of theamino acid pattern indicated in SEQ ID NO: 57, 58, 59, 60, 61, 62 or 63.

Further suitable nucleic acid sequences can be found by the skilledworker from the literature or the well-known gene libraries such as, forexample, ncbi.nlm.nih.gov.

In a further preferred embodiment of the process, additionally one ormore nucleic acid sequences which code for a polypeptide having theactivity of an ω-3-desaturase and/or of a Δ4-desaturase are introducedinto the plant.

The nucleic acid sequence which codes for a polypeptide having anω-3-desaturase activity is preferably selected from the group consistingof:

a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 193or 195, preferably the sequence depicted in SEQ ID NO: 193,

b) nucleic acid sequences which code for the amino acid sequenceindicated in SEQ ID NO: 194, c) nucleic acid sequences which hybridizewith the complementary strand of the nucleic acid sequence indicated inSEQ ID NO: 193 or 195 under stringent conditions, and

d) nucleic acid sequences which are at least 60%, 65%, 70%, 75% or 80%,preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%,particularly preferably at least 91%, 92%, 93%, 94% or 95%, andespecially at least 96%, 97%, 98% or 99%, identical to the sequenceindicated in SEQ ID NO: 193 or 195.

The ω-3-desaturase advantageously used in the process of the inventionmakes it possible to shift from the ω-6 biosynthetic pathway to the ω-3biosynthetic pathway, leading to a shift from C18:2 to C18:3 fattyacids. It is further advantageous for the ω-3-desaturase to convert awide range of phospholipids such as phosphatidylcholine (=PC),phosphatidylinositol (=PIS) or phosphatidylethanolamine (=PE). Finally,desaturation products can also be found in the neutral lipids (=NL),that is to say in the triglycerides.

The nucleic acid sequence which codes for a polypeptide having aΔ4-desaturase activity is preferably selected from the group consistingof:

a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 77,79, 81, 83, 85, 87, 89, 91 or 93, preferably having the sequencedepicted in SEQ ID NO: 77,

b) nucleic acid sequences which code for the amino acid sequenceindicated in SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, 92 or 94, preferablyfor the amino acid sequence indicated in SEQ ID NO: 78,

c) nucleic acid sequences which hybridize with the complementary strandof the nucleic acid sequences indicated in a) or b) above, especially ofthe nucleic acid sequence indicated in SEQ ID NO: 77, under stringentconditions,

d) nucleic acid sequences which are at least 60%, 65%, 70%, 75% or 80%,preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%,particularly preferably at least 91%, 92%, 93%, 94% or 95% andespecially at least 96%, 97%, 98% or 99%, identical to the sequenceindicated in SEQ ID NO: 77, and

e) nucleic acid sequences which code for an amino acid sequence whichhave at least one, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14, preferably all of the amino acid pattern indicated in SEQ ID NO: 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

The Δ4-desaturase which is advantageously used in the process of theinvention catalyzes the introduction of a double bond into the fattyacid docosapentaenoic acid, leading to formation of docosahexaenoicacid.

It is advantageous for the described process of the inventionadditionally to introduce further nucleic acids which code for enzymesof fatty acid or lipid metabolism into the plants in addition to thenucleic acid sequences which code for polypeptides having aΔ6-desaturase activity, a Δ6-elongase activity, a Δ5-desaturase activityand a Δ5-elongase activity, and to the nucleic acid sequences which areintroduced if appropriate and which code for a polypeptide having anω-3-desaturase activity and/or a Δ4-desaturase activity.

It is possible in principle to use all genes of fatty acid or lipidmetabolism in combination with the nucleic acid sequences used in theprocess of the invention; genes of fatty acid or lipid metabolismselected from the group of acyl-CoA dehydrogenase(s), acyl-ACP (acylcarrier protein) desaturase(s), acyl-ACP thioesterase(s), fatty acidacyltransferase(s),acyl-CoA:lysophospholipid acyltransferases, fattyacid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, alleneoxide synthases, hydroperoxide lyases or fatty acid elongase(s) arepreferably used in combination with the Δ6-elongase, Δ6-desaturase,Δ5-desaturase and the Δ5-elongase, and if appropriate the ω3-desaturaseand/or the Δ4-desaturase, it being possible to use individual genes or aplurality of genes in combination.

The nucleic acids used in the process of the invention areadvantageously expressed in vegetative tissues (somatic tissue).Vegetative tissue means in the context of this invention a tissue whichis propagated through mitotic divisions. Tissue of this type also arisesthrough asexual reproduction (apomixis) and propagation. Propagation isthe term used when the number of individuals increases in consecutivegenerations. These individuals arising through asexual propagation arevery substantially identical to their parents. Examples of such tissuesare leaf, flower, root, stalk, runners above or below ground (sideshoots, stolons), rhizomes, buds, tubers such as root tubers or stemtubers, bulb, brood bodies, brood buds, bulbuls or turion. Such tissuesmay also arise through pseudo vivipary, true vivipary or vivipary causedby humans. However, seeds arising through agamospermy, as are typical ofAsteraceae, Poaceae or Rosaceae, are also included among the vegetativetissues in which expression advantageously takes place. The nucleicacids used in the process of the invention are expressed to a smallextent or not at all in generative tissue (germ line tissue). Examplesof such tissues are tissues arising through sexual reproduction, i.e.meiotic cell divisions, such as, for example, seeds arising throughsexual processes.

A small extent means that, compared with vegetative tissue, theexpression measured at the RNA and/or protein level is less than 5%,advantageously less than 3%, particularly advantageously less than 2%,most preferably less than 1; 0.5; 0.25 or 0.125%.

The nucleic acid sequences are particularly preferably expressed in theleaves of the transgenic plants. This has the advantage that the LCPUFAsproduced according to the invention can be taken in by animals andhumans directly by consuming the leaves, and no previous processing ofthe plant material is necessary.

Expression of the nucleic acid sequences of the invention in the leafcan be achieved by using constitutive or leaf-specific promoters.

“Constitutive promoters” are promoters which make expression possible ina large number of, preferably in all, tissues over a substantial periodduring plant development, preferably throughout plant development. Apromoter from a plant or from a plant virus is preferably used. Thepromoter of the CaMV (cauliflower mosaic virus) 35S transcript (Francket al. (1980) Cell 21: 285-294), the 19S CaMV promoter (U.S. Pat. No.5,352,605), the actin promoter from rice (McElroy et al. (1990) PlantCell 2: 163-171), the legumin B promoter (GenBank Acc. No. X03677), theagrobacterium nopaline synthase promoter, the TR dual promoter, theagrobacterium octopine synthase promoter, the ubiquitin promoter(Holtorf et al. (1995) Plant Mol. Biol. 29: 637-649), the Smas promoter,the cinnamoyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),the promoters of the vacuolar ATPase subunits, the pEMU promoter (Lastet al. (1991) Theor. Appl. Genet. 81: 581-588), the MAS promoter (Veltenet al. (1984) EMBO J. 3(12): 2723-2730), the histone H3 promoter fromcorn (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285), the promoterof the nitrilase 1 gene from Arabidopsis (GenBank Acc. No. U38846,nucleotides 3862-5325) and the promoter of a proline-rich protein fromwheat (WO 91/13991) and further promoters which mediate constitutivegene expression. The promoter of the CaMV 35S transcript is particularlypreferred.

It is in principle possible to use all naturally occurring constitutivepromoters with their regulatory sequences like those mentioned above forthe novel process. However, it is likewise possible to use syntheticpromoters in addition or alone.

“Leaf-specific promoters” are promoters which show a high activity inthe leaf and no or only low activity in other tissues. “Low activity”means in the context of the invention that the activity in other tissuesis less than 20%, preferably less than 10%, particularly preferably lessthan 5% and most preferably less than 3, 2 or 1% of the activity in theleaf. Examples of suitable leaf-specific promoters are the promoters ofthe small subunit of rubisco (Timko et al. (1985) Nature 318: 579-582)and of the chlorophyll a/b-binding protein (Simpson et al. (1985) EMBOJ. 4: 2723-2729).

The skilled worker is aware of further leaf-specific promoters, or hecan isolate further suitable promoters with known methods. Thus, theskilled worker is able to identify leaf-specific regulatory nucleic acidelements with the aid of conventional methods of molecular biology, e.g.hybridization experiments or DNA-protein binding studies. This entailsfor example in a first step isolating the total poly(A)⁺ RNA from leaftissue of the desired organism from which the regulatory sequences areto be isolated, and setting up a cDNA library. In a second step, cDNAclones which are based on poly(A)⁺ RNA molecules from a non-leaf tissueare used to identify, by means of hybridization, those clones from thefirst library whose corresponding poly(A)⁺ RNA molecules accumulate onlyin leaf tissue. Subsequently, these cDNAs identified in this way areused to isolate promoters which have leaf-specific regulatory elements.Further PCR-based methods for isolating suitable leaf-specific promotersare additionally available to the skilled worker.

It is, of course, also possible for the nucleic acid sequences of thepresent invention to be expressed in the seeds of the transgenic plantsby using seed-specific promoters which are active in the embryo and/orin the endosperm. Seed-specific promoters can in principle be isolatedboth from dicotyledonous and from monocotyledonous plants. Preferredpromoters are listed hereinafter: USP (unknown seed protein) and vicilin(Vicia faba) (Bäumlein et al. (1991) Mol. Gen Genet. 225(3): 459-467),napin (oilseed rape) (U.S. Pat. No. 5,608,152), conlinin (flax) (WO02/102970), acyl-carrier protein (oilseed rape) (U.S. Pat. No. 5,315,001and WO 92/18634), oleosin (Arabidopsis thaliana) (WO 98/45461 and WO93/20216), phaseolin (Phaseolus vulgaris) (U.S. Pat. No. 5,504,200),Bce4 (WO 91/13980), legume B4 (LegB4 promoter) (Bäumlein et al. (1992)Plant J. 2(2): 233-239), Lpt2 and lpt1 (barley) (WO 95/15389 and WO95/23230), seed-specific promoters from rice, corn and wheat (WO99/16890), Amy32b, Amy 6-6 and aleurain (U.S. Pat. No. 5,677,474), Bce4(oilseed rape) (U.S. Pat. No. 5,530,149), glycinin (soybean) (EP 571741), phosphoenolpyruvate carboxylase (soybean) (JP 06/62870), ADR 12-2(soybean) (WO 98/08962), isocitrate lyase (oilseed rape) (U.S. Pat. No.5,689,040) or α-amylase (barley) (EP 781 849).

In a particularly preferred embodiment of the present invention, thenucleic acid sequences used, especially the nucleic acid sequence whichcodes for a Δ5-elongase and which is modified by comparison with thenucleic acid sequence in the organism from which the sequence originatesby being adapted to the codon usage in one or more plant species,preferably the nucleic acid sequence described in SEQ ID NO: 64, areexpressed in generative tissue, especially in the seed. Specificexpression in the seed advantageously takes place by using one of theabovementioned seed-specific promoters, especially using the napinpromoter. In this particularly preferred embodiment, the content ofproduced LCPUFAs, especially of the C22 fatty acids, in the seed oil isat least 5% by weight, advantageously at least 6, 7, 8, 9 or 10% byweight, preferably at least 11, 12, 13, 14 or 15% by weight,particularly preferably at least 16, 17, 18, 19 or 20% by weight, veryparticularly preferably at least 25, 30, 35 or 40% by weight, of theseed oil content. In a further particularly preferred embodiment withthe nucleic acid sequence described in SEQ ID NO: 63, the content of C22fatty acids in the seed oil is at least 8% by weight of the seed oilcontent.

In a further particularly preferred embodiment of the present invention,the nucleic acid sequences used, especially the nucleic acid sequencewhich codes for a Δ5-elongase and which is modified by comparison withthe nucleic acid sequence in the organism from which the sequenceoriginates by being adapted to the codon usage in one or more plantspecies, preferably the nucleic acid sequence described in SEQ ID NO:64, are expressed in generative tissue, especially in the seed. Specificexpression in the seed advantageously takes place by using one of theabovementioned seed-specific promoters, especially using the napinpromoter. In this particularly preferred embodiment, the content ofdocosahexaenoic acid in the seed oil is at least 1% by weight,preferably at least 1.1, 1.2, 1.3, 1.4 or 1.5% by weight, particularlypreferably at least 1.6, 1.7, 1.8 or 1.9% by weight, especially at least2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9% by weight, furtherpreferably at least 3, 3.5 or 4% by weight of the seed oil content. In afurther particularly preferred embodiment with the nucleic acid sequencedescribed in SEQ ID NO: 63, the content of docosahexaenoic acid in theseed oil is at least 1.9% by weight of the seed oil content. It is knownto the skilled worker in this connection that to produce docosahexaenoicacid additionally one or more nucleic acid sequences which codes for apolypeptide having the activity of a Δ4-desaturase activity arerequired. A nucleic acid sequence which codes for a polypeptide havingthe activity of a Δ4-desaturase activity is advantageously selected fromthe group consisting of nucleic acid sequences having the sequencedepicted in SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, 91 or 93, preferablyhaving the sequence depicted in SEQ ID NO: 77.

In a further particularly preferred embodiment of the present invention,the nucleic acid sequences used, especially the nucleic acid sequencewhich codes for a Δ5-elongase and which is modified by comparison withthe nucleic acid sequence in the organism from which the sequenceoriginates by being adapted to the codon usage in one or more plantspecies, preferably the nucleic acid sequence described in SEQ ID NO:64, are expressed in generative tissue, especially in the seed. Specificexpression in the seed advantageously takes place by using one of theabovementioned seed-specific promoters, especially using the napinpromoter. In this particularly preferred embodiment, the content ofdocosahexaenoic acid in the seed oil is at least 1% by weight,preferably at least 1.1, 1.2, 1.3, 1.4 or 1.5% by weight, particularlypreferably at least 1.6, 1.7, 1.8 or 1.9% by weight, especially at least2, 2.1, 2.2, 2.5, 2.6, 2.7, 2.8 or 2.9% by weight, further preferably atleast 3, 3.5 or 4% by weight of the seed oil content. In this case, thecontent of the produced LCPUFAs, especially of the C22 fatty acids, inthe seed oil is at least 5% by weight, advantageously at least 6, 7, 8,9 or 10% by weight, preferably at least 11, 12, 13, 14 or 15% by weight,particularly preferably at least 16, 17, 18, 19 or 20% by weight, veryparticularly preferably at least 25, 30, 35 or 40% by weight of the seedoil content. In a further particularly preferred embodiment with thenucleic acid sequence described in SEQ ID NO: 63, the content ofdocosahexaenoic acid in the seed oil is at least 1.9% by weight of theseed oil content, with the content of C22 fatty acids in the seed oilbeing at least 8% by weight of the seed oil content.

Plant gene expression can also be achieved via a chemically induciblepromoter (see a review in Gatz (1997) Annu. Rev. Plant Physiol. PlantMol. Biol., 48:89-108). Chemically inducible promoters are particularlysuitable when it is desired that the gene expression takes place in atime-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter.

Promoters which respond to biotic or abiotic stress conditions are alsosuitable, for example the pathogen-induced PRP1 gene promoter (Ward etal., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible tomatohsp80 promoter (U.S. Pat. No. 5,187,267), the chill-inducible potatoalpha-amylase promoter (WO 96/12814) or the wound-inducible pinIIpromoter (EP-A-0 375 091).

Other promoters which are also particularly suitable are those whichbring about the plastid-specific expression, since plastids constitutethe compartment in which precursors and some end products of lipidbiosynthesis are synthesized. Suitable promoters, such as the viral RNApolymerase promoter, are described in WO 95/16783 and WO 97/06250, andthe Arabidopsis clpP promoter, described in WO 99/46394.

It will be appreciated that the polyunsaturated fatty acids producedaccording to the invention can be produced not only in intact transgenicplants but also in plant cell cultures or in callous cultures.

The polyunsaturated fatty acids produced in the process areadvantageously bound in phospholipids and/or triacylglycerides, but mayalso occur as free fatty acids or else bound in the form of other fattyacid esters in the organisms. They may in this connection be present as“pure products” or else advantageously in the form of mixtures ofvarious fatty acids or mixtures of different phospholipids such asphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamineand/or phosphatidylserine and/or triacylglycerides, monoacyl-glyceridesand/or diacylglycerides. The LCPUFAs EPA, DPA and DHA produced in theprocess are advantageously present in phosphatidylcholine and/orphosphatidylethanolamine and/or in the triacylglycerides. Thetriacylglycerides may additionally also comprise further fatty acidssuch as short-chain fatty acids having 4 to 6 C atoms, medium-chainfatty acids having 8 to 12 C atoms or long-chain fatty acids having 14to 24 C atoms. They preferably comprise long-chain fatty acids,particularly preferably C20 or C22 fatty acids.

The term “glyceride” is understood as meaning glycerol esterified withone, two or three carboxyl radicals (mono-, di- or triglyceride).“Glyceride” is also understood as meaning a mixture of variousglycerides. The glyceride is preferably a triglyceride. The glyceride orglyceride mixture can comprise further additions, for example free fattyacids, antioxidants, proteins, carbohydrates, vitamins and/or othersubstances.

A “glyceride” for the purposes of the process according to the inventionis furthermore understood as meaning derivatives which are derived fromglycerol. In addition to the above-described fatty acid glycerides,these also include glycerophospholipids and glyceroglycolipids.Preferred examples which may be mentioned here are theglycerophospholipids such as lecithin (phosphatidylcholine),cardiolipin, phosphatidylglycerol, phosphatidylserine andalkylacylglycerophospholipids.

Phospholipids are to be understood as meaning, for the purposes of theinvention, phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol and/or phosphatidylinositol.

The fatty acid esters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fattyacid molecules can be isolated in the form of an oil or lipid, forexample in the form of compounds such as sphingolipids,phosphoglycerides, lipids, glycolipids such as glycosphingolipids,phospholipids such as phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol ordiphosphatidylglycerol, monoacylglycerides, diacylglycerides,triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters which comprise the polyunsaturated fatty acids with at leasttwo, three or four, preferably four, five or six double bonds, from theuseful plants which have been used for the preparation of the fatty acidesters; advantageously, they are isolated in the form of theirdiacylglycerides, triacylglycerides and/or in the form of thephosphatidyl ester, especially preferably in the form of thetriacylglycerides, phosphatidylcholine and/or phosphatidylethanolamine.In addition to these esters, the polyunsaturated fatty acids are alsopresent in the plants as free fatty acids or bound in other compounds.As a rule, the various abovementioned compounds (fatty acid esters andfree fatty acids) are present in the organisms with an approximatedistribution of 80 to 90% by weight of triglycerides, 2 to 5% by weightof diglycerides, 5 to 10% by weight of monoglycerides, 1 to 5% by weightof free fatty acids, 2 to 8% by weight of phospholipids, the total ofthe various compounds amounting to 100% by weight.

The LCPUFAs produced in the process of the invention are produced with acontent of at least 4% by weight, advantageously of at least 5, 6, 7, 8,9 or 10% by weight, preferably of at least 11, 12, 13, 14 or 15% byweight, particularly preferably of at least 16, 17, 18, 19, or 20% byweight, very particularly preferably of at least 25, 30, 35 or 40% byweight based on the total fatty acids in the transgenic plant. The fattyacids EPA, DPA and/or DHA produced in the process of the invention aremoreover present with a content of in each case at least 5% by weight,preferably of in each case at least 6, 7, 8 or 9% by weight,particularly preferably of in each case at least 10, 11 or 12% byweight, most preferably of in each case at least 13, 14, 15, 16, 17, 18,19 or 20% by weight based on the total fatty acids in the transgenicplant.

The fatty acids are advantageously produced in bound form. It ispossible with the aid of the nucleic acids used in the process of theinvention for these unsaturated fatty acids to be put on the sn1, sn2and/or sn3 position of the advantageously produced triacylglycerides.Advantageously, at least 11% of the triacylglycerides are doublysubstituted (meaning on the sn1 and sn2 or sn2 and sn3 positions).Triply substituted triacylglycerides are also detectable. Since aplurality of reaction steps take place from the starting compoundslinoleic acid (C18:2) and linolenic acid (C18:3), the final products ofthe process, such as, for example, arachidonic acid (ARA) oreicosapentaenoic acid (EPA), do not result as absolute pure products;traces or larger amounts of the precursors are always also present inthe final product. If, for example, both linoleic acid and linolenicacid are present in the initial plant, the final products such as ARA orEPA and/or DPA and/or DHA are also present as mixtures. The precursorsshould advantageously amount to not more than 20% by weight, preferablynot more than 15% by weight, particularly preferably not as 10% byweight, very particularly preferably not more than 5% by weight based onthe amount of the respective final product. Advantageously, only ARA orEPA and/or DPA and/or DHA are produced in the process of the invention,bound or as free acids, as final products in a transgenic plant.

Fatty acid esters or fatty acid mixtures produced by the process of theinvention advantageously comprise 6 to 15% palmitic acid, 1 to 6%stearic acid; 7-85% oleic acid; 0.5 to 8% vaccenic acid, 0.1 to 1%archaic acid, 7 to 25% saturated fatty acids, 8 to 85% monounsaturatedfatty acids and 60 to 85% polyunsaturated fatty acids, in each casebased on 100% and on the total fatty acid content of the organisms.Preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1%arachidonic acid in the total fatty acid content, are present asadvantageous polyunsaturated fatty acid in the fatty acid ester or fattyacid mixtures. The fatty acid esters or fatty acid mixtures produced bythe process of the invention further advantageously comprise fatty acidsselected from the group of fatty acids erucic acid (13-docosaenoicacid), sterculic acid (9,10-methyleneoctadec-9-enonic acid), malvalicacid (8,9-methyleneheptadec-8-enonic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienonic acid), vernonic acid(9,10-epoxyoctadec-12-enonic acid), taric acid (6-octadecynonic acid),6-nonadecynonic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynonic acid, pyrulic acid (t10-heptadecen-8-ynonic acid),crepenynic acid (9-octadecen-12-ynonic acid) 13,14-dihydrooropheic acid,octadecen-13-ene-9,11-diynonic acid, petroselenic acid(cis-6-octadecenonic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid, catalpic acid(9t11t13c-octadecatrienoic acid), eleosteric acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid) pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienonic acid). Ingeneral, the aforementioned fatty acids are advantageously present onlyin traces in the fatty acid esters or fatty acid mixtures produced bythe process of the invention, meaning that their occurrence, based onthe total fatty acid content, is less than 30%, preferably less than25%, 24%, 23%, 22% or 21%, particularly preferably less than 20%, 15%,10%, 9%, 8%, 7%, 6% or 5%, very particularly preferably less than 4%,3%, 2% or 1%. In a further preferred form of the invention theoccurrence of these aforementioned fatty acids, based on the total fattyacids, is less than 0.9%; 0.8%; 0.7%; 0.6% or 0.5%, particularlypreferably less than 0.4%; 0.3%; 0.2%; 0.1%. The fatty acid esters orfatty acid mixtures produced by the process of the inventionadvantageously comprise less than 0.1% based on the total fatty acidsand/or no butyric acid, no cholesterol and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

It is possible through the nucleic acid sequences used in the process ofthe invention to achieve an increase in the yield of LCPUFAs in thetransgenic plants of at least 50%, advantageously of at least 80%,particularly advantageously of at least 100%, very particularlyadvantageously of at least 150%, compared with the non-transgenicplants.

Chemically pure polyunsaturated fatty acids or fatty acid compositionscan also be synthesized by the processes described above. To this end,the fatty acids or the fatty acid compositions are isolated from theplants in the known manner, for example via extraction, distillation,crystallization, chromatography or a combination of these methods. Thesechemically pure fatty acids or fatty acid compositions are advantageousfor applications in the food industry sector, the cosmetic sector andespecially the pharmacological industry sector.

In principle, all dicotyledonous or monocotyledonous useful plants aresuitable for the process of the invention. Useful plants mean plantswhich serve to produce foods for humans and animals, to produce otherconsumables, fibers and pharmaceuticals, such as cereals, e.g. corn,rice, wheat, barley, millet, oats, rye, buckwheat; such as tubers, e.g.potato, cassava, sweet potato, yams etc.; such as sugar plants e.g.sugarcane or sugarbeet; such as legumes, e.g. beans, peas, broad beanetc.: such as oil and fat crops, e.g. soybean, oilseed rape, sunflower,safflower, flax, camolina etc., to mention only a few. Advantageousplants are selected from the group of plant families consisting of thefamilies of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae,Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae,Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreacea,Elaeagnaceae, Ericceae, Euphorbiaceae, Fabaceae, Fagaceae,Grossulariaceae, Juglandaceae, Lauraceae, Liliaceae, Linaceae,Malvaceae, Moraceae, Musaceae, Oleaceae, Oxalidaceae, Papaveraceae,Poaceae, Polygonaceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae,Scrophulariaceae, Solanaceae, Sterculiaceae and Valerianaceae.

Examples which may be mentioned are the following plants: Anacardiaceaesuch as the genera Pistacia, Mangifera, Anacardium, for example thegenus and species Pistacia vera [pistachio], Mangifer indica (mango) orAnacardium occidentale (cashew), Asteraceae such as the generaCalendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca,Locusta, Tagetes, Valeriana, e.g. the genus and species Calendulaofficinalis (common marigold), Carthamus tinctorius (safflower),Centaurea cyanus (cornflower), Cichorium intybus (chicory), Cynarascolymus (artichoke), Helianthus annus (sunflower), Lactuca sativa,Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa,Lactus scariola L. var. integrate, Lactuca scariola L. var.integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta (lettuce), Tagetes lucida, Tagetes erecta or Tagetes tenuifolia(French marigold), Apiaceae such as the genus Daucus, e.g. the genus andspecies Daucus carota (carrot), Betulaceae such as the genus Corylus,e.g. the genera and species Corylus avellana or Corylus colurna(hazelnut), Boraginaceae such as the genus Borago, e.g. the genus andspecies Borago officinalis (borage), Brassicaceae such as the generaBrassica, Camelina, Melanosinapis, Sinapis, Arabadopsis, e.g. the generaand species Brassica napus, Brassica rapa ssp. (oilseed rape), Sinapisarvensis Brassica juncea, Brassica juncea var. juncea, Brassica junceavar. crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Camelina sativa, Melanosinapis communis (mustard), Brassicaoleracea (feed beet) or Arabidopsis thaliana, Bromeliaceae such as thegenera Anana, Bromelia (pineapple), e.g. the genera and species Ananacomosus, Ananas ananas or Bromelia comosa (pineapple), Caricaceae suchas the genus Carica such as the genus and species Carica papaya(papaya), Cannabaceae such as the genus Cannabis such as the genus andspecies Cannabis sative (hemp), Convolvulaceae such as the generaIpomoea, Convolvulus, e.g. the genera and species Ipomoea batatus,Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoeafastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus(sweet potato, batate), Chenopodiaceae such as the genus Beta such asthe genera and species Beta vulgaris, Beta vulgaris var. altissima, Betavulgaris var. vulgaris, Beta maritima, Beta vulgaris var. perennis, Betavulgaris var. conditiva or Beta vulgaris var. esculenta (sugarbeet),Cucurbitaceae such as the genus Cucubita, e.g. the genera and speciesCucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata(pumpkin), Elaeagnaceae such as the genus Elaeagnus, e.g. the genus andspecies Olea europaea (olive), Ericaceae such as the genus Kalmia, e.g.the genera and species Kalmia latifolia, Kalmia angustifolia, Kalmiamicrophylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida (mountain laurel), Euphorbiaceae suchas the genera Manihot, Janipha, Jatropha, Ricinus, e.g. the genera andspecies Manihot utilissima, Janipha manihot, Jatropha manihot, Manihotaipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihotesculenta (cassava) or Ricinis communis (castor oil plant), Fabaceaesuch as the genera Pisum, Albizia, Cathormion, Feuillea, Inga,Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus,Soja, e.g. the genera and species Pisum sativum, Pisum arvense, Pisumhumile (pea), Albizia berteriana, Albizia julibrissin, Albizia lebbeck,Acacia berteriana, Acacia littoralis, Albizia berteriana, Albiziaberteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans,Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobiumberterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu,Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosaspeciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla,Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa(acacia), Medicago sativa, Medicago falcata, Medicago varia (alfalfa),Glycine max, Dolichos soja, Glycine gracilis, Glycine hispida, Phaseolusmax, Soja hispida or Soja max (soybean), Geraniaceae such as the generaPelargonium, Cocos, Oleum, e.g. the genera and species Cocos nucifera,Pelargonium grossularioides or Oleum cocois (coconut), Gramineae such asthe genus Saccharum, e.g. the genus and species Saccharum officinarum,Juglandaceae such as the genera Juglans, Wallia, e.g. the genera andspecies Juglans regia, Juglans ailanthifolia, Juglans sieboldiana,Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica,Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major,Juglans macrocarpa, Juglans nigra or Wallia nigra (walnut), Lauraceaesuch as the genera Persea, Laurus, e.g. the genera and species Laurusnobilis (bay), Persea americana, Persea gratissima or Persea persea(avocado), Leguminosae such as the genus Arachis. e.g. the genus andspecies Arachis hypogaea (peanut), Linaceae such as the genera Linum,Adenolinum, e.g. the genera and species Linum usitatissimum, Linumhumile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense or Linum trigynum (flax), Lythrarieae such asthe genus Punica, e.g. the genus and species Punica granatum(pomegranate), Malvaceae such as the genus Gossypium, e.g. the generaand species Gossypium hirsutum, Gossypium arboreum, Gossypiumbarbadense, Gossypium herbaceum or Gossypium thurberi (cotton), Musaceaesuch as the genus Musa, e.g. the genera and species Musa nana, Musaacuminata, Musa paradisiaca, Musa spp. (banana), Onagraceae such as thegenera Camissonia, Oenothera, e.g. the genera and species Oenotherabiennis or Camissonia brevipes (evening primrose), Palmae such as thegenus Elaeis, e.g. the genus and species Elaeis guineensis (oil palm),Papaveraceae such as the genus Papaver, e.g. the genera and speciesPapaver orientale, Papaver rhoeas, Papaver dubium (poppy), Pedaliaceaesuch as the genus Sesamum e.g the genus and species Sesamum indicum(sesame), Piperaceae such as the genera Piper, Artanthe, Peperomia,Steffensia, e.g. the genera and species Piper aduncum, Piper amalago,Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piperlongum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artantheelongata, Peperomia elongata, Piper elongatum, Steffensia elongata(cayenne pepper), Poaceae such as the genera Hordeum, Secale, Avena,Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (corn), Triticum, e.g.the genera and species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras,Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeumsativum, Hordeum secalinum (barley), Secale cereale (rye), Avena sativa,Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida(oats), Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghumvulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum, Panicum militaceum (millet), Oryzasativa, Oryza latifolia (rice), Zea mays (corn), Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare (wheat), Porphyridiaceae such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, e.g. the genus and species Porphyridiumcruentum, Proteaceae such as the genus Macadamia, e.g. the genus andspecies Macadamia intergrifolia (macadamia), Rubiaceae such as the genusCoffea, e.g. the genera and species Coffea spp., Coffea arabica, Coffeacanephora or Coffea liberica (coffee), Scrophulariaceae such as thegenus Verbascum, e.g. the genera and species Verbascum blattaria,Verbascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus (mullein), Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon, e.g. the genera and species Capsicumannuum, Capsicum annuum var. glabriusculum, Capsicum frutescens(pepper), Capsicum annuum (paprika), Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris (tobacco), Solanum tuberosum (potato),Solanum melongena (aubergine), Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum (tomato), Sterculiaceae such as the genus Theobroma, e.g.the genus and species Theobroma cacao (cocoa), or Theaceae such as thegenus Camellia, e.g. the genus and species Camellia sinensis (tea).

In an advantageous embodiment of the process, the useful plants used areoil fruit plants which comprise large amounts of lipid compounds, suchas peanut, oilseed rape, canola, sunflower, safflower (Carthamustinctoria), poppy, mustard, hemp, castor-oil plant, olive, sesame,Calendula, Punica, evening primrose, verbascum, thistle, wild roses,hazelnut, almond, macadamia, avocado, bay, pumpkin/squash, flax,soybean, pistachios, borage, trees (oil palm, coconut or walnut) orarable crops such as maize, wheat, rye, oats, triticale, rice, barley,cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato,tobacco, egg plant and tomato, Vicia species, pea, alfalfa or bushyplants (coffee, cacao, tea), Salix species, and perennial grasses andfodder crops. Advantageous plants according to the invention are oilfruit plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, flax, soybean, borage, trees (oilpalm,coconut). Especially preferred are plants which are high in C18:2-and/or C18:3-fatty acids, such as sunflower, safflower, tobacco,verbascum, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, flax, hemp or thistle. Very especially preferred plants areplants such as safflower, sunflower, poppy, evening primrose, walnut,flax, or hemp.

It is also advantageous to express the nucleic acid sequences of theinvention in the leaves of feed or food plants and thus to increase thecontent of eicosapentaenoic acid, docosapentaenoic acid and/ordocosahexaenoic acid in the leaves. Preferred feed plants are, forexample, trefoil species such as red clover (Trifolium pratense), whiteclover (Trifolium repens), alsike clover (Trifolium hybridum), sainfoin(Onobrychis viccifolia), Egyptian clover (Trifolium alexandrinium) andPersian clover (Trifolium resupinatum). Preferred food plants are forinstance lettuce species such as Lactuca sativa, Lactuca crispa, Lactucaesculenta, Lactuca scariola L. ssp. sativa, Lactuca scariola L. var.integrate, Lactuca scariola L. var. integrifolia, Lactuca sativa subsp.romana, Locusta communis and Valeriana locusta.

It is possible through the enzymatic activity of the nucleic acidsequences which are used in the process of the invention and which codefor polypeptides having Δ6-elongase, Δ6-desaturase, Δ5-desaturase and/orΔ5-elongase activity, advantageously in combination with nucleic acidsequences which code for polypeptides having ω3-desaturase and/orΔ44-desaturase activity, and further nucleic acid sequences which codefor polypeptides of fatty acid or lipid metabolism, such as furtherpolypeptides having Δ5-, Δ6-, Δ8-, Δ12-desaturase or Δ5-, Δ6- orΔ9-elongase activity, to produce a wide variety of polyunsaturated fattyacids in the process of the invention. Depending on the useful plantschosen for use in the process of the invention, mixtures of the variouspolyunsaturated fatty acids or individual polyunsaturated fatty acidssuch as EPA, DPA or DHA can be produced in free or bound form. Dependingon the prevailing fatty acid composition in the starting plant (C18:2 orC18:3 fatty acids), the resulting fatty acids are derived from C18:2fatty acids, such as GLA, DGLA or ARA or are derived from C18:3 fattyacids, such as EPA, DPA or DHA. If the only unsaturated fatty acidpresent in the plant used for the process is linoleic acid (LA,C18:2^(Δ9,12)), the only possible products of the process are GLA, DGLAand ARA, which may be present as free fatty acids or bound. If the onlyunsaturated fatty acid present in the plant used in the process isa-linolenic acid (ALA, C18:3^(Δ9,12,15)), for example as in flax, theonly possible products of the process are SDA, ETA, EPA, DPA and/or DHA,which may be present as described above as free fatty acids or bound. Itis possible to produce in a targeted manner only individual products inthe plant by modifying the activity of the enzymes used in the processand involved in the synthesis Δ6-elongase, Δ6-desaturase, Δ5-desaturaseand/or Δ6-elongase, advantageously in combination with further genes oflipid or fatty acid metabolism. Advantageously, only EPA, DPA or DHA ormixtures thereof are synthesized. Since the fatty acids are synthesizedin biosynthesis chains, the respective final products are not present aspure substances in the organisms. Small amounts of the precursorcompounds are always also present in the final product. These smallamounts are less than 20% by weight, advantageously less than 15% byweight, particularly advantageously less than 10% by weight, veryparticularly advantageously less than 5, 4, 3, 2 or 1% by weight basedon the final products EPA, DPA or DHA or mixtures thereof.

To increase the yield in the process according to the invention for theproduction of oils and/or triglycerides with a polyunsaturated fattyacid, content which is advantageously increased, it is advantageous toincrease the amount of starting product for the synthesis of fattyacids. This can be achieved for example by introducing a nucleic acidwhich encodes a polypeptide with 412-desaturase into the organism. Thisis particularly advantageous in useful plants, such as oil-producingplants such as plants of the Brassicaceae family, such as the genusBrassica, for example rape; the Elaeagnaceae family, such as the genusElaeagnus, for example the genus and species Olea europaea or the familyFabaceae, such as the genus Glycine, for example the genus and speciesGlycine max, which are high in oleic acid. Since these organisms have anonly low linoleic acid content (Mikoklajczak et al. (1961) Journal ofthe American Oil Chemical Society 38: 678-681) it is advantageous to usesaid Δ12-desaturases for producing the starting material linolenic acidfrom oleic acid. It is also possible in addition for the starting fattyacids to be provided from outside, but this is less preferred forreasons of cost.

Mosses and algae are the only plant systems known to produceconsiderable amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoicacid (DHA). Mosses comprise PUFAs in membrane lipids, whereas algae,organisms related to algae, and some fungi also accumulate substantialamounts of PUFAs in the triacylglycerol fraction. Nucleic acid moleculesisolated from strains which accumulate PUFAs also in the triacylglycerolfraction are therefore particularly advantageous for the process of theinvention and thus for modifying the lipid and PUFA production system ina plant such as a useful plant such as an oil crop plant, for exampleoilseed rape, canola, flax, hemp, soybean, sunflower, borage. They cantherefore advantageously be used in the process of the invention.

Nucleic acids used in the process of the invention are advantageouslyderived from plants such as algae, for example algae of the family ofPrasinophyceae such as from the genera Heteromastix, Mammella,Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus,Prasinococcus, Pseudoscourfielda, Pycnoocus, Pyramimonas, Scherffelia orTetraselmis such as the genera and species Heteromastix longifillis,Mamiella gilva, Mantoiella squamata, Micromonas pusilla, Nephroselmisolivacea, Nephroselmis pyriformis, Neproselmis rotunda, Ostreococcustauri, Ostreococcus sp. Prasinocladus ascus, Prasinocladus lubricus,Pycnococcus provasolii, Pyramimonas amylifera, Pyramimonas disomata,Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas parkae,Pyramimonas spinefera, Pyramimonas sp., Tetraselmis apiculta,Tetraselmis carteriaformis, Tetraselmis chui, Tetraselmis convolutae,Tetraselmis desikacharyi, Tetraselmis gracilis, Tetraselmis hazeni,Tetraselmis impellucida, Tetraselmis inconspicua, Tetraselmis levis,Tetraselmis maculata, Tetraselmis marina, Tetraselmis striata,Tetraselmis subcordiformis, Tetraselmis suecica, Tetraselmistetrabrachia, Tetraselmis tetrathele, Tetraselmis verrucosa, Tetraselmisverrucosa fo. rubens or Tetraselmis sp. or algae from the familyEuglenacease such as from the genera Ascoglena, Astasia, Colacium,Cyclidiopsis, Euglena, Euglenopsis, Hyalophacus, Khawkinea, Lepocinclis,Phacus, Strombomonas or Trachelomonas such as the genera and speciesEuglena acus, Euglena geniculata, Euglena gracilis, Euglenamixocylindracea, Euglena rostrifera, Euglena viridis, Colaciumstentorium, Trachelomonas cylindrica or Trachelomonas volvocina.

Further advantageous plants are algae such as Isochrysis orCrypthecodinium, algae/diatoms such as Thalassiosira or Phaeodactylum,mosses such as Physcomitrella or Ceratodon or higher plants such as thePrimulaceae such as Aleuritia, Calendula stella, Osteospermum spinescensor Osteospermum hyoseroides, microorganisms such as fungi such asAspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor orMortierella, bacteria such as shewanella, yeasts or animals such asnematodes such as Caenorhabditis, insects, frogs, sea cucumbers orfishes. The nucleic acid sequences isolated according to the inventionare advantageously derived from an animal from the order of vertebrates.The nucleic acid sequences are preferably derived from the class ofVertebrata; Euteleostomi, Actinopterygii; Neopterygii; Teleostei;Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae orOncorhynchus or Vertebrata, Amphibia, Anura, Pipidae, Xenopus orEvertebrata such as Protochordata, Tunicata, Holothuroidea, Cionidaesuch as Amaroucium constellatum, Botryllus schlosseri, Cionaintestinalis, Molgula citrina, Molgula manhattensis, Perophora viridisor Styela partita. The nucleic acids are particularly advantageouslyderived from fungi, animals or from plants such as algae or mosses,preferably from the order of Salmoniformes such as of the family ofSalmonidae such as of the genus Salmo, for example from the genera andspecies Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario, fromalgae such as the genera Mantoniella or Ostreococcus or from the diatomssuch as the genera Thalassiosira or Phaeodactylum or from algae such asCrypthecodinium.

In a preferred embodiment, the process further comprises the step ofobtaining a cell or a whole plant which comprises the nucleic acidsequences which are used in the process and which code for aΔ6-desaturase, Δ6-elongase, Δ5-desaturase and/or Δ5-elongase and, ifappropriate, nucleic acid sequences which code for an ω3-desaturaseand/or a Δ4-desaturase, it being possible for the cell and/or the usefulplant also to comprise further nucleic acid sequences of lipid or fattyacid metabolism. The nucleic acid sequences preferably used in theprocess are for expression advantageously incorporated into at least onegene construct and/or a vector as described hereinafter, alone or incombination with further nucleic acid sequences which code for proteinsof fatty acid or lipid metabolism, and finally transformed into the cellor plant. In a further preferred embodiment, this process furthercomprises the step of obtaining the oils, lipids or free fatty acidsfrom the useful plants, The cell produced in this way or the usefulplant produced in this way is advantageously a cell of an oil-producingplant, vegetable plant, lettuce plant, or ornamental plant or the plantitself as stated above.

Growing means for the cultivation in the case of plant cells, tissue ororgans on or in a nutrient medium or of the whole plant on or in asubstrate, for example in hydroculture, flower pot soil or on an arablefield.

For the purposes of the invention, “transgenic” or “recombinant” meanswith regard to, for example, a nucleic acid sequence, an expressioncassette (=gene construct) or a vector comprising the nucleic acidsequences used in the process according to the invention or a planttransformed with the nucleic acid sequences, expression cassette orvector used in the process according to the invention, all thoseconstructions brought about by recombinant methods in which either

-   a) the nucleic acid sequence, or-   b) a genetic control sequence which is operably linked with the    nucleic acid sequence, for example a promoter, or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by recombinant methods, it being possible for the    modification to be, for example, a substitution, addition, deletion,    inversion or insertion of one or more nucleotide residues. Natural    genetic environment means the natural genomic or chromosomal locus    in the original organism or the presence in a genomic library. In    the case of a genomic library, the natural genetic environment of    the nucleic acid sequence is preferably retained, at least in part.    The environment flanks the nucleic acid sequence at least on one    side and has a sequence length of at least 50 bp, preferably at    least 500 bp, especially preferably at least 1000 bp, very    especially preferably at least 5000 bp. A naturally occurring    expression cassette—for example the naturally occurring combination    of the natural promoter of the nucleic acid sequence used in the    process according to the invention with the nucleic acid sequence    which encodes proteins with corresponding Δ6-desaturase,    Δ6-elongase, Δ5-desaturase and Δ5-elongase activity, advantageously    in combination with nucleic acid sequences which encode proteins    having ω3-desaturase and/or Δ4-desaturase activity—becomes a    transgenic expression cassette when this expression cassette is    modified by non-natural, synthetic (“artificial”) methods such as,    for example, mutagenic treatment. Suitable methods are described,    for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

A “transgenic plant” for the purposes of the invention is understood asmentioned above as meaning that the nucleic acids used in the processare not at their natural locus in the genome of the plant. In this case,it is possible for the nucleic acid sequences to be expressedhomologously or heterologously. However, transgenic also means that,while the nucleic acids according to the invention are at their naturalposition in the genome of the plant, the sequence has been modified withregard to the natural sequence, and/or that the regulatory sequences ofthe natural sequences have been modified. Transgenic is preferablyunderstood as meaning the expression of the nucleic acids used in theprocess according to the invention at an unnatural locus in the genome,i.e. homologous or, preferably, heterologous expression of the nucleicacid sequences takes place.

Preferred transgenic organisms are useful plants such as oil-producingplants, vegetable plants, lettuce plants or ornamental plants which areadvantageously selected from the group of plant families consisting ofthe families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae,Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae,Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae,Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae,Grossulariaceae, Juglandaceae, Lauraceae, Liliaceae, Linaceae,Malvaceae, Moraceae, Musaceae, Oleaceae, Oxalidaceae, Papaveraceae,Poaceae, Polygonaceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae,Scrophulariaceae, Solanaceae, Sterculiaceae and Valerianaceae.

Host plants which are suitable for the nucleic acids, the expressioncassette or the vector used in the process according to the inventionare, in principle, advantageously all useful plants which are capable ofsynthesizing fatty acids, specifically unsaturated fatty acids, andwhich are suitable for the expression of recombinant genes. Exampleswhich should be mentioned at this point are plants such as Arabidopsis,Asteraceae such as Calendula or useful plants such as soybean, peanut,castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut,oil palm, safflower (Carthamus tinctorius) or cacao bean. Furtheradvantageous plants are mentioned at other points in this application.

Microorganisms are generally used as intermediate hosts for theproduction of transgenic useful plants. Such utilizable intermediatehost cells are detailed in: Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

Expression strains which can advantageously be used for this purposeare, for example, those with a lower protease activity. They aredescribed, for example, in: Gottesman, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128.

Transgenic plants which comprise the polyunsaturated, long-chain fattyacids synthesized in the process according to the invention canadvantageously be marketed directly without there being any need for theoils, lipids or fatty acids synthesized to be isolated. This form ofmarketing is particularly advantageous.

“Plants” for the purposes of the present invention are intact plants andall plant parts, plant organs or plant parts such as leaf, stem, seeds,root, tubers, anthers, fibers, root hairs, stalks, embryos, calli,cotelydons, petioles, harvested material, plant tissue, reproductivetissue and cell cultures which are derived from the actual transgenicplant and/or can be used for bringing about the transgenic plant. Inthis context, the seed comprises all parts of the seed such as the seedcoats, epidermal cells, seed cells, endosperm or embryonic tissue.

The compounds produced in the process of the invention can, however,also be isolated from the plants in the form of their oils, fat, lipidsand/or free fatty acids. Polyunsaturated fatty acids produced by theprocess of the invention can be obtained by harvesting the plants orplant cells either from the culture in which they grow or from thefield. This can take place by pressing or extracting the plant parts,preferably the plant seeds. It is possible in this connection for theoils, fats, lipids and/or free fatty acids to be obtained by pressing byso-called cold drawing or cold pressing without input of heat. To makeit easier to break open the plant parts, specifically the seeds, theyare previously crushed, steamed or roasted. The seeds pretreated in thisway can then be pressed or extracted with solvent such as warm hexane.The solvent is then removed again. It is possible in this way to isolatemore than 96% of the compounds produced in the process of the invention.The products obtained in this way are then processed further, that is tosay refined. This entails initially for example the plant mucilage andsuspended matter being removed. So-called desliming can take placeenzymatically or, for example, chemically/physically by adding acid suchas phosphoric acid. The free fatty acids are then removed by treatmentwith a base, for example sodium hydroxide solution. The resultingproduct is thoroughly washed with water to remove the alkali remainingin the product, and is dried. In order to remove the coloring mattersstill present in the product, the products are subjected to a bleachingwith, for example, bleaching earth or activated carbon. Finally, theproduct is also deodorized for example with steam.

The PUFAs or LCPUFAs produced by this process are preferably C20 and/orC22 fatty acid molecules having at least four double bonds in the fattyacid molecule, preferably five or six double bonds. These C20 and/or C22fatty acid molecules can be isolated from the plant in the form of anoil, lipid or a free fatty acid. Suitable transgenic plants are forexample those mentioned above.

These oils, lipids or fatty acids of the invention comprise, asdescribed above, advantageously 6 to 15% palmitic acid, 1 to 6% stearicacid; 7-85% oleic acid; 0.5 to 8% vaccenic acid, 0.1 to 1% archaic acid,7 to 25% saturated fatty acids, 8 to 85% monounsaturated fatty acids and60 to 85% polyunsaturated fatty acids, in each case based on 100% and onthe total fatty acid content of the plants.

Advantageous polyunsaturated, long-chain fatty acids present in thefatty acid esters or fatty acid mixtures such as phosphatidyl fatty acidesters or triacylglyceride esters are preferably at least 10; 11; 12;13; 14; 15; 16; 17; 18; 19 or 20% by weight based on the total fattyacid content of eicosapentaenoic acid, based on the total fatty acidcontent, and/or at least 1; 2; 3; 4; 5 or 6% by weight ofdocosapentaenoic acid, based on the total fatty acid content, and/or atleast 1; 2; 3; preferably at least 4; 5; 6; particularly preferably atleast 7 or 8 and most preferably at least 9 or 10% by weight ofdocosahexaenoic acid, based on the total fatty acid content.

The fatty acid esters or fatty acid mixtures which have been produced bythe process of the invention further comprise fatty acids selected fromthe group of fatty acids erucic acid (13-docosaic acid), sterculic acid(9,10-methylene octadec-9-enonic acid), malvalic acid (8,9-methyleneheptadec-8-enonic acid), chaulmoogrinic acid (cyclopentenedodecanoicacid), furan fatty acid (9,12-epoxyoctadeca-9,11-dienonoic acid),vernonoic acid (9,10-epoxyoctadec-12-enonoic acid), tarinic acid(6-octadecynonic acid), 6-nonadecynonic acid, santalbic acid(t11-octadecen-9-ynoic acid), 6,9-octadecenynonic acid, pyrulic acid(t10-heptadecen-8-ynonic acid), crepenynic acid (9-octadecen-12-ynonicacid) 13,14-dihydrooropheic acid, octadecen-13-ene-9,11-diynonic acid,petroselenic acid (cis-6-octadecenonic acid), 9c,12t-octadecadienoicacid, calendulic acid (8t10t12c-octadecatrienoic acid, catalpic acid(9t11t13c-octadecatrienoic acid), eleosteric acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid) pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienonic acid). Ingeneral, the aforementioned fatty acids are advantageously present onlyin traces in the fatty acid esters or fatty acid mixtures produced bythe process of the invention, meaning that their occurrence, based onthe total fatty acid content, is less than 30%, preferably less than25%, 24%, 23%, 22% or 21%, particularly preferably less than 20%, 15%,10%, 9%, 8%, 7%, 6% or 5%, very particularly preferably less than 4%,3%, 2% or 1%. In a further preferred form of the invention theoccurrence of these aforementioned fatty acids, based on the total fattyacids, is less than 0.9%; 0.8%; 0.7%; 0.6% or 0.5%, particularlypreferably less than 0.4%; 0.3%; 0.2%; 0.1%. The fatty acid esters orfatty acid mixtures produced by the process of the inventionadvantageously comprise less than 0.1% based on the total fatty acidsand/or no butyric acid, no cholesterol and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

A further embodiment according to the invention is the use of the oils,the lipids, the fatty acids and/or the fatty acid composition, which areproduced by the process of the invention, in feeding stuffs, foodstuffs,cosmetics or pharmaceuticals. The oils, lipids, fatty acids or fattyacid mixtures obtained in the process according to the invention can beused for admixture with other oils, lipids, fatty acids or fatty acidmixtures of animal origin, such as, for example, fish oils, in themanner with which the skilled worker is familiar. These oils, lipids,fatty acids or fatty acid mixtures which are produced in this way andconsist of vegetable and animal components can also be used for thepreparation of feeding stuffs, foodstuffs, cosmetics or pharmaceuticals.

The term “oil”, “lipid” or “fat” is understood as meaning a fatty acidmixture comprising unsaturated and/or saturated, preferably esterifiedfatty acid(s). It is preferred that the oil, fat or lipid is high inpolyunsaturated free or advantageously esterified fatty acid(s), inparticular linoleic acid, y-linolenic acid, dihomo-y-linolenic acid,arachidonic acid, a-linolenic acid, stearidonic acid, eicosatetraenoicacid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoicacid. Preferably, the amount of unsaturated esterified fatty acids isapproximately 30%, with an amount of 50% being especially preferred andan amount of 60%, 70%, 80% or more being most preferred. The amount ofthe fatty acid can be determined by gas chromatography after convertingthe fatty acids into the methyl esters by transesterification. The oil,lipid or fat can comprise various other saturated or unsaturated fattyacids, for example calendulic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid and the like. In particular, the amount of thevarious fatty acids can vary, depending on the starting plant.

As described above, the polyunsaturated fatty acid esters advantageouslyhaving three, four, five or six, particularly advantageously having fiveor six double bonds and which have been prepared in the processadvantageously take the form of fatty acid esters, for example,sphingolipid esters, phosphoglyceride esters, lipid esters, glycolipidesters, phospholipid esters, monoacylglycerol esters, diacylglycerolesters, triacylglycerol esters or other fatty acid esters, preferencebeing given to phospholipid esters and/or triacylglycerol esters.

Starting with the polyunsaturated fatty acid esters produced thus in theprocess according to the invention and advantageously having at leastthree, four, five or six double bonds, the polyunsaturated fatty acidswhich are present can be liberated for example via treatment withalkali, for example with aqueous KOH or NaOH, or by acid hydrolysis,advantageously in the presence of an alcohol such as methanol orethanol, or via enzymatic cleavage, and isolated via, for example, phaseseparation and subsequent acidification with, for example, H₂SO₄.However, the fatty acids can also be liberated directly without theabove-described processing steps.

Substrates of the nucleic acid sequences used in the process whichencode polypeptides with Δ6-desaturase, Δ6-elongase, Δ5-desaturaseand/or Δ5-elongase activity and optionally nucleic acid sequences whichencode polypeptides having ω3-desaturase and/or Δ4-desaturase activity,and/or of the further nucleic acids which are used, such as the nucleicacid sequences which encode polypeptides of the fatty acid or lipidmetabolism selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyl transferase(s),acyl-CoA:lysophospholipid acyltransferases, fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenases, lipoxygenases, triacylglycerol lipases, allenoxidesynthases, hydroperoxide lyases or fatty acid elongase(s) areadvantageously C₁₆-, C₁₈- or C₂₀-fatty acids. Preferably, the fattyacids converted in the process as substrates are converted in the formof their acyl-CoA esters and/or in the form of their phospholipidesters.

To produce the long-chain PUFAs according to the invention, thesaturated, monounsaturated C₁₆-fatty acids and/or polyunsaturatedC₁₈-fatty acids must first, depending on the substrate, be desaturatedand/or elongated or only deaturated by the enzymatic activity of adesaturase and/or elongase and subsequently elongated by at least twocarbon atoms by an elongase. After one elongation cycle, this enzymeactivity leads either starting from C₁₆-fatty acids to C₁₈-fatty acidsor starting from C₁₈-fatty acids to C₂₀-fatty acids, and after twoelongation cycles starting from C₁₆-fatty acids leads to C₂₀-fattyacids. The activity of the desaturases or elongases used in the processaccording to the invention preferably leads to C₂₀- and/or C₂₂-fattyacids, advantageously with at least two or three double bonds in thefatty acid molecule, preferably with four, five or six double bonds,especially preferably to C₂₂-fatty acids with at least five double bondsin the fatty acid molecule. Especially preferred products of the processaccording to the invention are eicosapentaenoic acid, docosapentaenoicacid and/or docosahexaenoic acid. The C₁₈-fatty acids with at least twodouble bonds in the fatty acid can be elongated by the enzymaticactivity according to the invention in the form of the free fatty acidor in the form of the esters, such as phospholipids, glycolipids,sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol ortriacylglycerol.

The preferred biosynthesis site of fatty acids, oils, lipids or fats inthe plants which are advantageously used is, for example, generally theseed or cell layers of the seed, so that seed-specific expression of thenucleic acids used in the process makes sense. However, it is obviousthat the biosynthesis of fatty acids, oils or lipids need not be limitedto the seed tissue, but may also take place in a tissue specific mannerin all of the remaining parts of the plant, for example in epidermalcells or in the tubers. The synthesis advantageously takes placeaccording to the inventive process in the vegetative (somatic) tissue.

Owing to the method according to the invention, the polyunsaturatedfatty acids which are produced can, in principle, be increased in twoways in the plants used in the process. Advantageously the pool of freepolyunsaturated fatty acids and/or the amount of the esterifiedpolyunsaturated fatty acids produced by the process can be increased.Advantageously, the pool of esterified polyunsaturated fatty acids inthe transgenic plants is increased by the process according to theinvention, advantageously in the form of the phosphatidyl esters and/ortriacyl esters.

The sequences used in the process of the invention are cloned singlyinto expression constructs or provided on a joint recombinant nucleicacid molecule and used for introduction and for expression in organisms.These expression constructs make it possible for the polyunsaturatedfatty acids produced in the process of the invention to be synthesizedoptimally.

The nucleic acids used in the process may, after introduction into aplant or plant cell, either be located on a separate plasmid oradvantageously be integrated into the genome of the host cell. In thecase of integration into the genome, the integration may be random ortake place by recombination such that the native gene is replaced by theintroduced copy, thus modulating production of the desired compound bythe cell, or through use of a gene in trans, so that the gene isfunctionally connected to a functional expression unit which comprisesat least one sequence ensuring the expression of a gene and at least onesequence ensuring the polyadenylation of a functionally transcribedgene. The nucleic acid sequences are advantageously introduced into theplants via multiexpression cassettes or constructs for multiparallelexpression, i.e. the nucleic acid sequences are present in a jointexpression unit.

The nucleic acid construct may comprise more than one nucleic acidsequence coding for a polypeptide having the enzymatic activity of aΔ12-desaturase, Δ4-desaturase, Δ5-desaturase, Δ6-desaturase,Δ5-elongase, Δ6-elongase, and/or ω3-desaturase. It is also possible fora plurality of copies of a nucleic acid sequence coding for apolypeptide having the enzymatic activity of a Δ12-desaturase,Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ5-elongase, Δ6-elongase,and/or ω3-desaturase to be present.

For the introduction, the nucleic acids used in the process areadvantageously amplified and ligated in the known manner. Preferably, aprocedure following the protocol for Pfu DNA polymerase or a Pfu/Taq DNApolymerase mixture is followed. The primers are selected depending onthe sequence to be amplified. The primers should expediently be chosenin such a way that the amplicon comprises the entire codogenic sequencefrom the start codon to the stop codon. After the amplification, theamplificon is expediently analyzed. For example, the analysis can becarried out by gel-electrophoretic separation with respect to qualityand quantity. Thereafter, the amplicon can be purified following astandard protol (for example Qiagen). An aliquot of the purifiedamplicon is then available for the subsequent cloning step. Suitablecloning vectors are generally known to the skilled worker. Theseinclude, in particular, vectors which are capable of replication inmicrobial systems, that is to say mainly vectors which ensure efficientcloning in yeasts or fungi and which make possible the stabletransformation of plants. Those which must be mentioned in particularare various binary and cointegrated vector systems which are suitablefor the T-DNA-mediated transformation. Such vector systems are, as arule, characterized in that they comprise at least the vir genesrequired for the Agrobacterium-mediated transformation and theT-DNA-delimiting sequences (T-DNA border). These vector systemspreferably also comprise further cis-regulatory regions such aspromoters and terminators and/or selection markers, by means of whichsuitably transformed organisms can be identified. While in the case ofcointegrated vector systems vir genes and T-DNA sequences are arrangedon the same vector, binary systems are based on at least two vectors,one of which bears vir genes, but no T-DNA, while a second one bearsT-DNA, but no vir genes. Owing to this fact, the last-mentioned vectorsare relatively small, easy to manipulate and capable of replication bothin E. coli and in Agrobacterium. These binary vectors include vectorsfrom the series pBIB-HYG, pPZP, pBecks, pGreen. In accordance with theinvention, pBin19, pBI101, pBinAR, pGPTV and pCAMBIA are used bypreference. An overview of the binary vectors and their use is found inHellens et al. (2000) Trends in Plant Science 5: 446-451.

In order to prepare the vectors, the vectors can first be linearizedwith restriction endonuclease(s) and then modified enzymatically in asuitable manner. Thereafter, the vector is purified, and an aliquot isemployed for the cloning step. In the cloning step, the enzymaticallycleaved and, if appropriate, purified amplificate is cloned with vectorfragments which have been prepared in a similar manner, using ligase. Inthis context, a particular nucleic acid construct, or vector or plasmidconstruct, can have one or more than one codogenic gene segments. Thecodogenic gene segments in these constructs are preferably linkedoperably with regulatory sequences. The regulatory sequences include, inparticular, plant sequences such as the above-described promoters andterminators. The constructs can advantageously be stably propagated inmicroorganisms, in particular Escherichia coli and Agrobacteriumtumefaciens, under selective conditions and thus make possible thetransfer of heterologous DNA into plants.

The nucleic acid sequences and nucleic acid constructs used in theinventive process can be introduced into microorganisms and then intoplants, advantageously using cloning vectors, and thus be used in thetransformation of plants such as those which are published in and citedtherein: Plant Molecular Biology and Biotechnology (CRC Press, BocaRaton, Fla.), Chapter 6/7, p. 71-119 (1993); F. F. White, Vectors forGene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1,Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993,15-38; B. Jenes et al., Techniques for Gene Transfer, in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. (1991) 42: 205-225. Thus, the nucleic acids, nucleicacid constructs and/or vectors used in the process can be used for therecombinant modification of a broad spectrum of plants so that thelatter become better and/or more efficient LCPUFA producers.

Owing to the introduction of a Δ6-desaturase, Δ6-elongase, Δ5-desaturaseand Δ5-elongase gene into a plant, alone or in combination with othergenes, it is not only possible to increase biosynthesis flux towards theend product, but also to increase, or to create de novo thecorresponding triacylglycerol and/or phosphatidylester composition.Likewise, the number or activity of other genes which are involved inthe import of nutrients which are required for the biosynthesis of oneor more fatty acids, oils, polar and/or neutral lipids, can beincreased, so that the concentration of these precursors, cofactors orintermediates within the cells or within the storage compartment isincreased, whereby the ability of the cells to produce PUFAs, asdescribed below, is enhanced further. By optimizing the activity orincreasing the number of one or more of the Δ6-desaturase, Δ6-elongase,Δ5-desaturase and/or Δ5-elongase genes which are involved in thebiosynthesis of these compounds, or by destroying the activity of one ormore genes which are involved in the degradation of these compounds, itmay be possible to increase the yield, production and/or productionefficiency in fatty acid and lipid molecules from organisms andadvantageously from plants.

The nucleic acid molecules used in the process of the invention code forproteins or parts thereof, whereas the proteins or the individualprotein or parts thereof comprises an amino acid sequence which hassufficient homology to an amino acid sequence which is depicted in thesequences SEQ ID NO: 65, SEQ ID NO: 2, SEQ ID NO: 172 or SEQ ID NO: 52and, if appropriate, SEQ ID NO: 194 or SEQ ID NO: 78, so that theproteins or parts thereof still have a Δ6-desaturase, Δ6-elongase,Δ5-desaturase and/or Δ5-elongase activity and, if appropriate, aΔ4-desaturase and/or ω3-desaturase activity. The proteins or partsthereof which is/are encoded by the nucleic acid molecule/nucleic acidmolecules preferably still have its/their essential enzymatic activityand the ability to participate in the metabolism of compounds necessaryfor constructing cell membranes or lipid bodies in organisms,advantageously in plants, or in the transport of molecules across thesemembranes. The proteins encoded by the nucleic acid molecules are atleast about 60% and preferably at least about 70%, 80% or 90%, andparticularly preferably at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequences depicted in SEQ ID NO: 65, SEQ ID NO: 2, SEQ ID NO:172, SEQ ID NO: 52, SEQ ID NO: 194 or SEQ ID NO: 78. Homology orhomologous means in the context of the invention identity or identical.

The homology was calculated over the entire amino acid or nucleic acidsequence region. To compare various sequences, the skilled worker hasavailable a series of programs which are based on various algorithms.The algorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. The program PileUp (J. Mol. Evolution(1987) 25: 351-360; Higgins et al. (1989) CABIOS 5: 151-153) or theprograms Gap and BestFit (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453 and Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489),which are part of the GCG software packet [Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711 (1990], were used to carry outthe sequence comparisons. The sequence homology data given above in %were determined over the entire sequence region using the program GAPwith the following settings: Gap Weight: 50, Length Weight: 3, AverageMatch: 10.000 and Average Mismatch: 0.000. Unless otherwise specified,these settings were always used as standard settings for sequencecomparisons.

Essential enzymatic activity of the ω3-desaturase, Δ6-desaturase,Δ6-elongase, Δ5-elongase, Δ4-desaturase and/or Δ5-desaturase used in theprocess of the invention means that, compared with the proteins/enzymesencoded by the sequence having SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO:171, SEQ ID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77, they still have anenzymatic activity of at least 10%, preferably of at least 20%,particularly preferably of at least 30% and most preferably of at least40, 50 or 60%, and thus are able to participate in the metabolism ofcompounds necessary for synthesizing fatty acids, advantageously fattyacid esters such as phosphatidyl esters and/or triacylglyceride esters,in a plant or plant cell, or in the transport of molecules acrossmembranes.

Nucleic acids which can be advantageously used in the process arederived from bacteria, fungi, diatoms, animals such as Caernorhabditisor Oncorhynchus or plants such as algae or mosses such as the generaShewanella, Physcomitrella, Thraustochytrium, Fusarium, Phytophthora,Ceratodon, Pytium irregulare, Mantoniella, Ostreococcus, Isochrysis,Aleurita, muscarioides, Mortierella, Borago, Phaeodactylum,Crypthecodinium, specifically from the genera and species Pytiumirregulare, Oncorhynchus mykiss, Xenopus laevis, Ciona intestinalis,Thalassiosira pseudonona, Mantoniella squamata, Ostreococcus sp.,Ostreococcus tauri, Euglena gracilis, Physcomitrella patens, Phytophtorainfestans, Fusarium graminaeum, Cryptocodinium cohnii, Ceratodonpurpureus, Isochrysis galbana, Aleurita farinosa, Thraustochytrium sp.,Muscarioides viallii, Mortierella alpina, Borago officinalis,Phaeodactylum tricornutum, Caenorhabditis elegans or particularlyadvantageously from Pytium irregulare, Thraustochytrium sp. and/orOstreococcus tauri.

It is possible additionally to use in the process of the inventionnucleotide sequences which code for a Δ12-desaturase, Δ9-elongase orΔ8-desaturase. The nucleic acid sequences used in the process areadvantageously introduced in an expression cassette which makesexpression of the nucleic acids in plants possible.

The nucleic acid sequences which code for the Δ12-desaturase,ω3-desaturase, Δ9-elongase, Δ6-desaturase, Δ8-desaturase, Δ6-elongase,Δ5-desaturase, Δ5-elongase or Δ4-desaturase are functionally linked toone or more regulatory signals to increase the gene expression. Theseregulatory sequences are intended to make targeted expression of thegenes possible. This may mean for example, depending on the plant, thatthe gene is expressed and/or overexpressed only after induction, or thatit is expressed and/or overexpressed immediately. Sequencesadvantageously used for the expression make constitutive expressionpossible, such as CaMV35S, CaMV36S, CaMV35Smas, nos, mas, ubi, stpt, leaor Super promoter. Expression preferably takes place in vegetativetissue as described above. In another preferred embodiment, theexpression takes place in seeds.

These regulatory sequences are for example sequences to which inducersor repressors bind and thus regulate the expression of the nucleic acid.In addition to the regulatory sequences which are not linked in theirnatural locus to the nucleic acid sequences, or instead of thesesequences, the natural regulation of these sequences may still bepresent before the actual structural genes and, if appropriate, havebeen genetically modified so that natural regulation is switched off andexpression of the genes is increased. The gene construct mayadditionally advantageously also comprise one or more so-called“enhancer sequences” functionally linked to the promoter, which makeincreased expression of the nucleic acid sequence possible. Additionaladvantageous sequences can also be inserted at the 3′ end of the DNAsequences, such as further regulatory elements or terminators.Advantageous terminators are for example viral terminators such as the35S terminator or others. The nucleic acid sequences used in the processaccording to the invention may be present in one or more copies of theexpression cassette (=gene construct). Preferably, only one copy of thegenes is present in each expression cassette. This gene construct, orthe gene constructs, can be introduced into the plant simultaneously orsuccessively and expressed together in the host organism. In thiscontext, the gene construct(s) can be inserted in one or more vectorsand be present in the cell in free form, or else be inserted in thegenome. It is advantageous for the insertion of further genes in theplant when the genes to be expressed are present together in one geneconstruct. However, it is also possible to introduce in each case onegene construct containing a nucleic acid sequence into a plant and tocross the resulting plants with one another in order to obtain progenywhich contains all gene constructs together.

In this context, the regulatory sequences or factors can, as describedabove, preferably have a positive effect on the gene expression of thegenes introduced, thus enhancing it. Thus, an enhancement of theregulatory elements, advantageously at the transcriptional level, maytake place by using strong transcription signals such as promotersand/or enhancers. In addition, however, enhanced translation is alsopossible, for example by improving the stability of the mRNA.

To ensure the stable integration of the biosynthesis genes into thetransgenic plant over a plurality of generations, each of the nucleicacids which encode Δ6-desaturase, Δ6-elongase, Δ5-desaturase orΔ5-elongase and if appropriate the ω3-desaturase or Δ4-desaturase andwhich are used in the process should be expressed under the control of aseparate promoter. This can be identical or different for each of thesequences. In this context, the expression cassette is advantageouslyconstructed in such a way that a promoter is followed by a suitablecleavage site for insertion of the nucleic acid to be expressed, whichcleavage site is advantageously in a polylinker. If appropriate, aterminator can be positioned behind the polylinker. This sequence isrepeated several times, preferably three, four, five or six times, sothat up to six genes can be combined in one construct and thusintroduced into the transgenic plant in order to be expressed. Toexpress the nucleic acid sequences, the latter are inserted behind thepromoter via the suitable cleavage site, for example in the polylinker.Advantageously, each nucleic acid sequence has its own promoter and, ifappropriate, its own terminator. However, it is also possible to inserta plurality of nucleic acid sequences behind a promoter and, ifappropriate, before a terminator.

Here, the insertion site, or the sequence, of the inserted nucleic acidsin the expression cassette is not of critical importance, that is to saya nucleic acid sequence can be inserted at the first or last position inthe cassette without the expression being substantially influenced bythe position. In an advantageous embodiment, different promoters suchas, for example, the USP, LegB4 or DC3 promoter, and differentterminators can be used in the expression cassette. In a furtheradvantageous embodiment, identical promoters such as the CaMV35Spromoter can also be used.

As described above, the transcription of the genes which have beenintroduced should advantageously be terminated by suitable terminatorsat the 3′ end of the biosynthesis genes which have been introduced(behind the stop codon). An example of a sequence which can be used inthis context is the OCS 1 or the 35SCaMV terminator. As is the case withthe promoters, different terminator sequences should be used here foreach gene.

As described above, the gene construct can also comprise further genesto be introduced into the organisms. It is possible and advantageous tointroduce into the host plants, and to express therein, regulatory genessuch as genes for inductors, repressors or enzymes which, owing to theirenzyme activity, engage in the regulation of one or more genes of abiosynthesis pathway. These genes can be of heterologous or ofhomologous origin. Moreover, further biosynthesis genes of the fattyacid or lipid metabolism can advantageously be present in the nucleicacid construct, or gene construct or alternatively, these genes can alsobe present on one further or more further nucleic acid constructs. Abiosynthesis gene of the fatty acid or lipid metabolism which ispreferably chosen is one or more genes selected from the group ofacyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl-transferase(s),acyl-CoA:lysophospholipid acyltransferases, fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenases, lipoxygenases, triacylglycerol lipases, allenoxidesynthases, hydroperoxide lyases or fatty acid elongase(s) orcombinations thereof. Especially advantageous nucleic acid sequences arebiosynthesis genes of the fatty acid or lipid metabolism selected fromthe group of the acyl-CoA:lysophospholipid acyltransferase,Δ8-desaturase, Δ9-desaturase, Δ12-desaturase and/or Δ9-elongase.

In this context, the abovementioned nucleic acids or genes can be clonedinto expression cassettes, like those mentioned above, in combinationwith other elongases and desaturases and used for transforming plantswith the aid of Agrobacterium.

The term “vector” used in this description relates to a nucleic acidmolecule which is capable of transporting another nucleic acid to whichit is bound. One type of vector is a “plasmid”, a circulardouble-stranded DNA loop into which additional DNA segments can beligated. A further type of vector is a viral vector, it being possiblefor additional DNA segments to be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey have been introduced (for example bacterial vectors with bacterialreplication origin). Other vectors are advantageously integrated intothe genome of a host cell when they are introduced into the host cell,and thus replicate together with the host genome. Moreover, certainvectors can govern the expression of genes with which they are inoperable linkage. These vectors are referred to in the present contextas “expression vectors”. Usually, expression vectors which are suitablefor DNA recombination techniques take the form of plasmids. In thepresent description, “plasmid” and “vector” can be used exchangeablysince the plasmid is the form of vector which is most frequently used.However, the invention is also intended to cover other forms ofexpression vectors, such as viral vectors, which exert similarfunctions. Furthermore, the term vector is also intended to encompassother vectors with which the skilled worker is familiar, such as phages,viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids,phagemids, cosmids, linear or circular DNA.

The recombinant expression vectors advantageously used in the processcomprise the nucleic acid sequences or the above-described geneconstruct used in the process in a form which is suitable for expressingthe nucleic acids used in a host cell, which means that the recombinantexpression vectors comprise one or more regulatory sequences, which areselected on the basis of the host cells to be used for the expression,which regulatory sequence(s) is/are linked operably with the nucleicacid sequence to be expressed. In a recombinant expression vector,“linked operably” means that the nucleotide sequence of interest isbound to the regulatory sequence(s) in such a way that the expression ofthe nucleotide sequence is made possible and they are bound to eachother in such a way that both sequences carry out the predicted functionwhich is ascribed to the sequence (for example in an in-vitrotranscription/translation system, or in a host cell if the vector isintroduced into the host cell). The term “regulatory sequence” isintended to comprise promoters, enhancers and other expression controlelements (for example polyadenylation signals). These regulatorysequences are described, for example, in Goeddel: Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990), or see: Gruber and Crosby, in: Methods in Plant MolecularBiology and Biotechnology, CRC Press, Boca Raton, Fla., Ed.: Glick andThompson, Chapter 7, 89-108, including the references cited therein.Regulatory sequences comprise those which govern the constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich govern the direct expression of the nucleotide sequence only inspecific host cells under specific conditions. The skilled worker knowsthat the design of the expression vector can depend on factors such asthe choice of host cell to be transformed, the desired degree ofexpression of the protein and the like.

The recombinant expression vectors used can be designed for theexpression of the nucleic acid sequences used in the process in such away that they can be transformed into prokaryotic intermediate hosts andfinally, after introduction into the plants, make expression of thegenes possible therein. This is advantageous because on account ofsimplicity, intermediate steps in vector construction are frequentlycarried out in microorganisms. For example, the Δ6-desaturates,Δ6-elongase, Δ5-desaturate and/or Δ5-elongase genes can be expressed inbacterial cells, insect cells (using baculovirus expression vectors),yeast cells and other fungal cells (see Romanos, M. A., et al. (1992)Yeast 8:423-488; van den Hondel, C. A. M. J. J., et al. (1991)“Heterologous gene expression in filamentous fungi”, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, Editors, pp.396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J.,& Punt, P. J. (1992) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F., et al., Editors, pp. 1-28, Cambridge University Press: Cambridge),Algae (Falciatore et al. (1999) Marine Biotechnology.1: (3):239-251),ciliates, with vectors following a transformation process as describedin WO 98/01572, and preferably in cells of multicellular plants (seeSchmidt, R. and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.:538-586; Plant Molecular Biology andBiotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer in:Transgenic Plants, vol. 1, Engineering and Utilization, Editors.: Kungand R. Wu, Academic Press (1993), 128-43; Potrykus (1991) Annu. Rev.Plant Physiol. Plant Molec. Biol. 42: 205-225 (and references citedtherein)). Suitable hosts are what are further discussed in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). The recombinant expression vector mayalternatively be transcribed and translated in vitro for example usingT7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes usually takes place with vectorswhich comprise constitutive or inducible promoters which control theexpression of fusion or non-fusion proteins. Typical fusion expressionvectors are inter alia pGEX (Pharmacia Biotech Inc; Smith, D. B., andJohnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), of whichglutathione S-transferase (GST), maltose E-binding protein and proteinA, respectively, are fused to the recombinant target protein.

Examples of suitable inducible non-fusion E. coli expression vectors areinter alia pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionby the pTrc vector is based on transcription by host RNA polymerase froma hybrid trp-lac fusion promoter. Target gene expression from the pET11d vector is based on transcription from a T7-gn10-lac fusion promoterwhich is mediated by a coexpressed viral RNA polymerase (T7 gn1). Thisviral polymerase is provided by the host strains BL21 (DE3) or HMS174(DE3) from a resident X prophage which harbors a T7 gn1 gene undertranscription control of the lacUV 5 promoter.

Other vectors suitable in prokaryotic organisms are known to the skilledworker; these vectors are for example in E. coli pLG338, pACYC184, thepBR series such as pBR322, the pUC series such as pUC18 or pUC19, theM113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, Xgtll or pBdCI, in Streptomyces pIJ101, pIJ364,pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667.

In a further embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in the yeast S. cerevisiaeinclude pYeDesaturasec1 (Baldari et al. (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al.(1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and processes for constructing vectors suitable for usein other fungi, such as the filamentous fungi, are described in detailin: van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of fungi, J. F. Peberdy et al., editors, pp. 1-28,Cambridge University Press: Cambridge, or in: More Gene Manipulations inFungi (J. W. Bennet & L. L. Lasure, Editors, pp. 396-428: AcademicPress: San Diego). Further suitable yeast vectors are for example pAG-1,YEp6, YEp13 or pEMBLYe23.

Alternatively, the nucleic acid sequences used in the process of theinvention can be expressed in insect cells using baculovirus expressionvectors. Baculovirus vectors available for expressing proteins incultured insect cells (e.g. Sf9 cells) include the pAc series (Smith etal. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

The above mentioned vectors provide only a small survey of possiblesuitable vectors. Further plasmids are known to the skilled worker andare described for example in: Cloning Vectors (Editors Pouwels, P. H. etal., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).Further suitable expression systems for prokaryotic and eukaryotic cellssee in chapters 16 and 17 of Sambrook, J., Fritsch, E. F., and Maniatis,T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold springHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

The genes used in the process can also be expressed in single-celledplant cells (such as algae), see Falciatore et al. (1999) MarineBiotechnology 1 (3):239-251 and references cited therein, and in plantcells from higher plants (for example spermatophytes such as arablecrops). Examples of plant expression vectors comprise those which aredescribed in detail in: Becker, D., Kemper, E., Schell, J., andMasterson, R. (1992) Plant Mol. Biol. 20:1195-1197; and Bevan, M. W.(1984) Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer inHigher Plants; in: Transgenic Plants, Vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38.

A plant expression cassette preferably comprises regulatory sequenceswhich are capable of governing the expression of genes in plant cellsand are linked operably so that each sequence can fulfil its function,such as transcriptional termination, for example polyadenylationsignals. Preferred polyadenylation signals are those which are derivedfrom Agrobacterium tumefaciens T-DNA, such as gene 3 of the Ti plasmidpTiACH5 (Gielen et al., (1984) EMBO J. 3 835 et seq.), which is known asoctopine synthase, or functional equivalents thereof, but all otherterminators which are functionally active in plants are also suitable.

Since the regulation of plant gene expression is very often not limitedto the transcriptional level, a plant expression cassette preferablycomprises other sequences which are linked operably, such as translationenhancers, for example the overdrive sequence, which enhances thetobacco mosaic virus 5′-untranslated leader sequence, which increasesthe protein/RNA ratio (Gallie et al. (1987) Nucl. Acids Research15:8693-8711).

As described above, the plant gene expression must be linked operablywith a suitable promoter which controls gene expression. Advantageouslyutilizable promoters are constitutive promoters (Benfey et al., EMBO J.(1989) 8: 2195-2202), such as those which are derived from plantviruses, such as 35S CaMV (Franck et al. (1980) Cell 21: 285-294), 19SCaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), or plantpromoters, such as the promoter of the Rubisco small subunit, which isdescribed in U.S. Pat. No. 4,962,028.

Other preferred sequences for use for functional connection in plantgene expression cassettes are targeting sequences which are necessaryfor guiding the gene product into its appropriate cellular compartment,for example into the vacuoles, the cell nucleus, all types of plastidssuch as amyloplasts, chloroplasts, chromoplasts, the extracellularspace, the mitochondria, the endoplasmic reticulum, oil bodies,peroxisomes and other compartments of plant cells; (see a review inKermode (1996) Crit. Rev. Plant Sci. 15(4): 284-423 and literature citedtherein).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viatraditional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction asused in the present context are intended to encompass the multiplicityof prior-art methods for introducing heterologous nucleic acids (forexample DNA) into a host cell, including calcium phosphate or calciumchloride coprecipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2^(nd) Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory manuals suchas Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,Ed: Gartland and Davey, Humana Press, Totowa, N.J.

The term “nucleic acid (molecule)” as used herein comprises in anadvantageous embodiment additionally the untranslated sequence locatedat the 3′ end and at the 5′ end of the coding gene region: at least 500,preferably 200, particularly preferably 100 nucleotides of the sequenceupstream of the 5′ end of the coding region and at least 100, preferably50, particularly preferably 20 nucleotides of the sequence downstream ofthe 3′ end of the coding gene region. An “isolated” nucleic acidmolecule is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. An “isolated” nucleicacid preferably has no sequences which naturally flank the nucleic acidin the genomic DNA of the organism from which the nucleic acid isderived (e.g. sequences located at the 5′ and 3′ ends of the nucleicacid). In various embodiments, the isolated Δ6-desaturase, Δ6-elongaseor Δ5-desaturase and, if appropriate, the ω3-desaturase or Δ4-desaturasemolecule used in the process may for example comprise less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequenceswhich naturally flank the nucleic acid molecule in the genomic DNA ofthe cell from which the nucleic acid is derived.

The nucleic acid molecules used in the process can be isolated by usingstandard techniques of molecular biology and the sequence informationprovided herein. It is also possible for example with the aid ofcomparative algorithms to identify a homologous sequence or homologous,conserved sequence regions at the DNA or amino acid level. These can beused as hybridization probe in standard hybridization techniques (asdescribed for example in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) forisolating further nucleic acid sequences useful in the process. Thenucleic acid molecule used in the process, or parts thereof, canmoreover be isolated by polymerase chain reaction, in which caseoligonucleotide primers based on this sequence or on parts thereof areused (e.g. a nucleic acid molecule comprising the complete sequence or apart thereof can be isolated by polymerase chain reaction usingoligonucleotide primers which have been constructed on the basis of thisidentical sequence). For example, mRNA can be isolated from cells (e.g.by the guanidinium thiocyanate extraction method of Chirgwin et al.(1979) Biochemistry 18:5294-5299) and cDNA can be prepared with the aidof reverse transcriptase (e.g. Moloney MLV reverse transcriptaseobtainable from Gibco/BRL, Bethesda, Md. or AMV reverse transcriptase,obtainable from Seikagaku America, Inc., St. Petersburg, Fla.).Synthetic oligonucleotide primers for amplification by means ofpolymerase chain reaction can be constructed on the basis of one of thesequences shown in SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO: 171, SEQ IDNO: 51, SEQ ID NO: 193 or SEQ ID NO: 77 or with the aid of the aminoacid sequences depicted in SEQ ID NO: 65, SEQ ID NO: 2, SEQ ID NO: 172,SEQ ID NO: 52, SEQ ID NO: 194 or SEQ ID NO: 78. A nucleic acid of theinvention can be amplified by standard PCR amplification techniquesusing cDNA or alternatively genomic DNA as template and suitableoligonucleotide primers. The nucleic acid amplified in this way can becloned into a suitable vector and characterized by DNA sequenceanalysis. Oligonucleotides can be prepared by standard syntheticmethods, for example using an automatic DNA synthesizer.

Homologs of the Δ5-elongase, ω3-desaturase, Δ6-desaturase, Δ6-elongase,Δ4-desaturase or Δ5-desaturase nucleic acid sequences used, having thesequence SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO: 171, SEQ ID NO: 51, SEQID NO: 193 or SEQ ID NO: 77, mean for example allelic variants having atleast about 40, 50 or 60%, preferably at least about 60 or 70%, morepreferably at least about 70 or 80%, 90% or 95% and even more preferablyat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity or homology to one of the nucleotidesequences shown in SEQ ID NO: 64, 66, 68 or 70, to one of the nucleotidesequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39 or 41, to one of the nucleotide sequencesshown in SEQ ID NO: 171, 173, 175, 177, 179, 181 or 183, to one of thenucleotide sequences shown in SEQ ID NO: 51, 53 or 55, to one of thenucleotide sequences shown in SEQ ID NO: 193 or 195 or to one of thenucleotide sequences shown in or SEQ ID NO: 77, 79, 81, 83, 85, 87, 89,91 or 93, especially the nucleotide sequences shown in SEQ ID NO: 64,SEQ ID NO: 1, SEQ ID NO: 171, SEQ ID NO: 51, SEQ ID NO: 193 or SEQ IDNO: 77, or their homologs, derivatives or analogs or parts thereof. Alsoincluded are isolated nucleic acid molecules of a nucleotide sequencewhich hybridize for example under stringent conditions to one of thenucleotide sequences shown in SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO:171, SEQ ID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77 or a part thereof. Apart means in this connection according to the invention that at least25 base pairs (=bp), 50 bp, 75 bp, 100 bp, 125 bp or 150 bp, preferablyat least 175 bp, 200 bp, 225 bp, 250 bp, 275 bp or 300 bp, particularlypreferably 350 bp, 400 bp, 450 bp, 500 bp or more base pairs are usedfor the hybridization. It is also possible advantageously to use thecomplete sequence. Allelic variants comprise in particular functionalvariants which can be obtained by deletion, insertion or substitution ofnucleotides from the sequence depicted in SEQ ID NO: 64, SEQ ID NO: 1,SEQ ID NO: 171, SEQ ID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77, butwhere the enzyme activity of the proteins encoded thereby issubstantially retained for the insertion.

Nucleic acid molecules advantageous for the process of the invention canbe isolated on the basis of their homology to the ω3-desaturase,Δ6-desaturase, Δ5-desaturase, Δ5-elongase, Δ4-desaturase and/orΔ6-elongase nucleic acid sequences disclosed herein by using thesequences or a part thereof as hybridization probe in standardhybridization techniques under stringent hybridization conditions. It ispossible in this connection for example to use isolated nucleic acidmolecules which are at least 15 nucleotides long and hybridize understringent conditions with the nucleic acid molecules which comprise anucleotide sequence of SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO: 171, SEQID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77. It is also possible to usenucleic acid molecules having at least 25, 50, 100, 250 or morenucleotides.

The term “hybridizes under stringent conditions” as used herein isintended to describe hybridization and washing conditions under whichnucleic acid sequences which are at least 60% mutually homologousnormally remain hybridized together. The conditions are preferably suchthat sequences which are at least about 65%, preferably at least about70% and particularly preferably at least about 75% or more mutuallyhomologous normally remain hybridized together. These stringentconditions are known to the skilled worker and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-restrictive example of stringenthybridization conditions are hybridizations in 6× sodium chloride/sodiumcitrate (=SSC) at about 45° C., followed by one or more washing steps in0.2×SSC, 0.1% SDS at 50 to 65° C. The skilled worker is aware that thesehybridization conditions differ depending on the type of nucleic acidand, for example organic solvents are present, in relation to thetemperature and the concentration of the buffer. The temperature forexample under “standard hybridization conditions” is, depending on thetype of nucleic acid, between 42° C. and 58° C. in aqueous buffer with aconcentration of 0.1 to 5×SSC (pH 7.2). If organic solvent, for example50% formamide, is present in the abovementioned buffer, the temperatureunder standard conditions is about 42° C. The hybridization conditionsfor DNA:DNA hybrids are preferably for example 0.1×SSC and 20° C. to 45°C., preferably 30° C. to 45° C. The hybridization conditions for DNA:RNAhybrids are preferably for example 0.1×SSC and 30° C. to 55° C.,preferably 45° C. to 55° C. The aforementioned hybridizationtemperatures are determined for example for a nucleic acid with a lengthof about 100 bp (=base pairs) and a G+C content of 50% in the absence offormamide. The skilled person knows how the necessary hybridizationconditions can be determined on the basis of textbooks such as theabovementioned or from the following textbooks Sambrook et al.,“Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (editors) 1985, “Nucleic Acids Hybridization: A PracticalApproach”, IRL Press at Oxford University Press, Oxford; Brown (editor)1991, “Essential Molecular Biology: A Practical Approach”, IRL Press atOxford University Press, Oxford.

In order to determine the percentage of homology (=identity) of twoamino acid sequences (for example one of the sequences of SEQ ID NO: 65,SEQ ID NO: 2, SEQ ID NO: 172, SEQ ID NO: 52, SEQ ID NO: 194 or SEQ IDNO: 78) or of two nucleic acids (for example SEQ ID NO: 64, SEQ ID NO:1, SEQ ID NO: 171, SEQ ID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77), thesequences are written one under the other in order to be able to comparethem optimally (for example, gaps may be introduced into the sequence ofa protein or of a nucleic acid in order to generate optimal alignmentwith the other protein or the other nucleic acid). Then, the amino acidradicals or nucleotides at the corresponding amino acid positions ornucleotide positions are compared. If a position in a sequence isoccupied by the same amino acid radical or the same nucleotide as thecorresponding position in another sequence, then the molecules arehomologous at this position (i.e. amino acid or nucleic acid “homology”as used in the present context corresponds to amino acid or nucleic acid“identity”). The percentage of homology between the two sequences is afunction of the number of identical positions which the sequences share(i.e. % homology=number of identical positions/total number of positions×100). The programs and algorithms used to determine the homology aredescribed above.

An isolated nucleic acid molecule which codes for an ω3-desaturase,Δ6-desaturase, Δ5-desaturase, Δ5-elongase, Δ4-desaturase and/orΔ6-elongase which is used in the process and which is homologous to aprotein sequence of SEQ ID NO: 65, SEQ ID NO: 2, SEQ ID NO: 172, SEQ IDNO: 52, SEQ ID NO: 194 or SEQ ID NO: 78 can be generated by introducingone or more nucleotide substitutions, additions or deletions into anucleotide sequence of SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO: 171, SEQID NO: 51, SEQ ID NO: 193 or SEQ ID NO: 77, so that one or more aminoacid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced into one of the sequencesof SEQ ID NO: 64, SEQ ID NO: 1, SEQ ID NO: 171, SEQ ID NO: 51, SEQ IDNO: 193 or SEQ ID NO: 77 by standard techniques such as site-specificmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions are preferably produced at one or more of the predictednonessential amino acid residues. In a “conservative amino acidsubstitution” the amino acid residue is replaced by an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g. lysine, arginine,histidine), acidic side chains (e.g. aspartic acid, glutamic acid),uncharged polar side chains (e.g. glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g. threonine,valine, isoleucine) and aromatic side chains (e.g. tyrosine,phenylalanine, tryptophan, histidine). A predicted nonessential aminoacid residue in an ω3-desaturase, Δ6-desaturase, Δ5-desaturase,Δ5-elongase, Δ4-desaturase or Δ6-elongase is thus preferably replaced byanother amino acid residue from the same side-chain family. Analternative possibility in another embodiment is to introduce themutations randomly over the whole or a part of the ω3-desaturase-,Δ6-desaturase-, Δ5-desaturase-, Δ5-elongase-, Δ4-desaturase- orΔ6-elongase-encoding sequence, e.g. by saturation mutagenesis, and theresulting mutants can be screened for the ω3-desaturase, Δ6-desaturase,Δ5-desaturase, Δ5-elongase, Δ4-desaturase or Δ6-elongase activitydescribed herein in order to identify mutants which have retained theω3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ5-elongase, Δ4-desaturaseor Δ6-elongase activity. The encoded protein can be recombinantlyexpressed after the mutagenesis, and the activity of the protein can bedetermined for example by using the assays described herein.

The invention is illustrated in greater detail by the examples whichfollow, which are not to be construed as limiting. The content of all ofthe references, patent applications, patents and published patentapplications cited in the present patent application is herewithincorporated by reference.

The following table shows the sequence identifiers as used in thepriority application of Feb. 21, 2006, with the German applicationnumber 102006008030.0, and the corresponding sequence identifiers inthis subsequent application. The nucleic acid sequence identified by SEQID NO: 1 of the priority application corresponds for example to thenucleic acid sequence identified by SEQ ID NO: 64 of the subsequentapplication.

Table of concordance of sequence identifiers of the priority applicationand the sequence identifiers in the subsequent application:

SEQ ID NO: Priority application SEQ ID NO: German application thisnumber subsequent 102006008030.0 application Organism 1 64 Ostreococcustauri 2 65 Ostreococcus tauri 3 1 Phytium irregulare 4 2 Phytiumirregulare 5 171 Traustochytrium sp. 6 172 Traustochytrium sp. 7 51Thraustochytrium ssp. 8 52 Thraustochytrium ssp. 9 193 Phytophthorainfestans 10 194 Phytophthora infestans 11 77 Traustochytrium sp. 12 78Traustochytrium sp. 13 109 Ostreococcus tauri n.a. 110 Ostreococcustauri 14 122 Ostreococcus tauri n.a. 123 Ostreococcus tauri 15 143Ostreococcus tauri 16 144 Ostreococcus tauri 17 161 Cauliflower mosaicvirus 18 162 Cauliflower mosaic virus 19 163 Thalassiosira pseudonana 20164 Thalassiosira pseudonana

EXAMPLES Example 1: General Cloning Methods

The cloning methods such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli cells, bacterial cultures and thesequence analysis of recombinant DNA were carried out as described bySambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Example 2: Sequence Analysis of Recombinant DNA

Recombinant DNA molecules were sequenced with an ABI laser fluorescenceDNA sequencer by the method of Sanger (Sanger et al. (1977) Proc. Natl.Acad. Sci. USA 74: 5463-5467). Fragments resulting from a polymerasechain reaction were sequenced and verified to avoid polymerase errors inconstructs to be expressed.

Example 3: Cloning of Genes from Ostreococcus tauri

It was possible by searching for conserved regions in an Ostreococcustauri sequence database (genomic sequences) in each case a sequencecoding for a protein having Δ5-elongase activity or Δ6-elongaseactivity. These are the following sequences:

Gene name SEQ ID Amino acids OtELO1.1, (Δ6-Elongase) SEQ ID NO: 143 292OtELO2.1, (Δ5-Elongase) SEQ ID NO: 109 300

OtElo2.1 shows greatest similarity to an elongase from Danio rerio(GenBank AAN77156; approx. 26% identity), whereas OtElo1.1 showsgreatest similarity to the elongase from Physcomitrella (PSE) (approx.36% identity) (alignments were carried out with the tBLASTn algorithm(Altschul et al. (1990) J. Mol. Biol. 215: 403-410)).

The cloning of the elongases was carried out as follows:

40 ml of an Ostreococcus tauri culture in the stationary phase were spundown and resuspended in 100 μl of double-distilled water and stored at−20° C. The corresponding genomic DNAs were amplified by the PCR method.The corresponding primer pairs were selected so that they harbored theyeast consensus sequence for high-efficiency translation (Kozak (1986)Cell 44: 283-292) beside the start codon. Amplification of the OtEloDNAs was carried out in each case with 1 μl of thawed cells, 200 μMdNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a totalvolume of 50 μl. The conditions for the PCR were as follows: firstdenaturation at 95° C. for 5 minutes, followed by 30 cycles at 94° C.for 30 seconds, 55° C. for 1 minute and 72° C. for 2 minutes, and afinal elongation step at 72° C. for 10 minutes.

Example 4: Optimization of Elongase Genes from Ostreococcus tauri

Elongases from the organism Ostreococcus tauri were isolated asdescribed in example 3. In order to achieve an increase in the contentof C22 fatty acids, the sequences SEQ ID NO: 143 (Δ6-elongase) and SEQID NO: 109 (coding for a protein identified by SEQ ID NO: 110)(Δ5-elongase) were adapted to the codon usage in oilseed rape, flax andsoybean. For this purpose, the amino acid sequence of the Δ6-elongaseand of the Δ5-elongase (SEQ ID NO: 144 for the Δ6-elongase; SEQ ID NO:65 for the Δ5-elongase) was back-translated to obtain degenerate DNAsequences. These DNA sequences were adapted by means of theGeneOptimizer program (from Geneart, Regensburg) to the codon usage inoilseed rape, soybean and flax, taking account of the natural frequencyof individual codons. The optimized sequences obtained in this way,which are indicated in SEQ ID NO: 64 (Δ5-elongase) and SEQ ID NO: 122(coding for a protein identified by SEQ ID NO: 123) (Δ6-elongase) weresynthesized in vitro.

Example 5: Cloning of Expression Plasmids for Heterologous Expression inYeasts

To characterize the function of the optimized nucleic acid sequences,the open reading frames of the respective DNAs were cloned downstream ofthe galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO(Invitrogen), resulting in the plasmids pOTE1.2 (comprising theΔ6-elongase sequence) and pOTE2.2 (comprising the Δ5-elongase sequence).

Overview of the elongase sequences cloned into the yeast vectorpYES2.1/V5-His-TOPO:

Gene name SEQ ID Amino acids pOTE1.1, (Δ6-elongase) SEQ ID NO: 143 292pOTE1.2, (Δ6-elongase) SEQ ID NO: 122 292, codon- optimized pOTE2.1,(Δ5-elongase) SEQ ID NO: 109 300 pOTE2.2, (Δ5-elongase) SEQ ID NO: 64300, codon- optimized

The Saccharomyces cerevisiae strain 334 was transformed byelectroporation (1500 V) with the vectors pOTE1.2 and pOTE2.2 and withthe comparative constructs pOTE1.1 and pOTE2.1 which comprise thenatural nucleic acid sequence coding for the Δ6-elongase andΔ5-elongase, respectively. A yeast transformed with the empty vectorpYES2 was used as control. The transformed yeasts were selected oncomplete minimal medium (CMdum) agar plates with 2% glucose but withouturacil. After the selection, three transformants in each case wereselected for further functional expression.

To express the Ot elongases, initially precultures composed of in eachcase 5 ml of CMdum liquid medium with 2% (w/v) raffinose but withouturacil were inoculated with the selected transformants and incubated at30° C., 200 rpm for 2 days. 5 ml of CMdum liquid medium (without uracil)with 2% raffinose were then inoculated with the precultures to an OD₆₀₀of 0.05. Moreover, 0.2 mM y-linolenic acid (GLA) was added in each caseto the yeast culture transformed with pOTE1.1 and pOTE1.2. On the basisof the activity of OtELO1.1, an elongation of the y-linolenic acid tothe 20:3 fatty acid is to be expected. Respectively 0.2 mM arachidonicacid and eicosapentaenoic acid were added in each case to the yeastculture transformed with pOTE2.1 and pOTE2.2. Corresponding to theactivity of OtELO2.1, it is to be expected that the fatty acids ARA andEPA will be elongated respectively to the 22:4 and 22:5 fatty acids.Expression was induced by adding 2% (w/v) galactose. The cultures wereincubated at 20° C. for a further 96 h.

Example 6: Expression of OtELO2.2 (as Depicted in SEQ ID NO: 64) andOtELO1.2 (as in SEQ ID NO: 122) in Yeasts

Yeasts transformed as in example 5 with the plasmids pYES2, pOTE1.2 andpOTE2.1 were analyzed in the following way:

The yeast cells from the main cultures were harvested by centrifugation(100×g, 5 min, 20° C.) and washed with 100 mM NaHCO₃, pH 8.0, in orderto remove remaining medium and fatty acids. Fatty acid methyl esters(FAMEs) were prepared from the yeast cell sediments by acidicmethanolysis. For this purpose, the cell sediments were incubated with 2ml of 1 N methanolic sulfuric acid and 2% (v/v) dimethoxypropane at 80°C. for 1 h. The FAMES were extracted by extraction twice with petroleumether (PE). To remove underivatized fatty acids, the organic phases werewashed once each with 2 ml of 100 mM NaHCO₃, pH 8.0 and with 2 ml ofdistilled water. The PE phases were then dried with Na₂SO₄, evaporatedunder argon and taken up in 100 μl of PE. The samples were separated ona DB-23 capillary column (30 m, 0.25 mm, 0.25 μm, Agilent) in aHewlett-Packard 6850 gas chromatograph with flame ionization detector.The conditions for the GLC analysis were as follows: the oventemperature was programmed from 50° C. to 250° C. at a rate of 5° C./minand finally 10 min at 250° C. (holding).

The signals were identified by comparing the retention times withappropriate fatty acid standards (Sigma). The methodology is describedfor example in Napier and Michaelson (2001) Lipids 36(8): 761-766;Sayanova et al. (2001) Journal of Experimental Botany 52(360):1581-1585, Sperling et al. (2001) Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al. (1998) FEBS Letters 439(3): 215-218. Theresults of the analyses are depicted in table 1.

It was possible to confirm the appropriate activities both forpOTE1.1/pOTE1.2 and for pOTE2.1/2.2. The optimized sequence(respectively pOTE1.2 and pOTE2.2) showed activity in both cases.Synthesis of y-linolenic acid could be increased only slightly bypOTE1.2 compared with the wild-type sequence. By contrast, it waspossible to observe for pOTE2.2 surprisingly both an increase in theactivity and an alteration in the specificity (table 1). In thisconnection, the activity for elongation of EPA had virtually doubled,while the elongation of ARA had more than quadrupled. It was thuspossible with the optimization of the sequence of the Δ5-elongase fromOstreococcus tauri to increase the yield of the precursors of DHA 6-foldin yeast with the same amount of substrate.

Example 7: Cloning Expression Plasmids for the Seed-Specific Expressionin Plants

The following general conditions described apply to all subsequentexperiments unless described otherwise.

pBin19, pBI101, pBinAR, pGPTV, pCAMBIA or pSUN are preferably used forthe following examples in accordance with the invention. An overview ofthe binary vectors and their use can be found in Hellens et al, Trendsin Plant Science (2000) 5: 446-451. A pGPTV derivative as described inDE10205607 was used. This vector differs from pGPTV by an additionallyinserted AscI restriction cleavage site.

Starting point of the cloning procedure was the cloning vector pUC19(Maniatis et al.). In the first step, the conlinin promoter fragment wasamplified using the following primers:

Cnl1 C 5′: (SEQ ID NO: 200)gaattcggcgcgccgagctcctcgagcaacggttccggcggtatagagttg ggtaattcgaCnl1 C 3′: (SEQ ID NO: 201)cccgggatcgatgccggcagatctccaccattttttggtggtgat

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme EcoRI and then for 12 hours at 25° C. with therestriction enzyme SmaI. The cloning vector pUC19 was incubated in thesame manner. Thereafter, the PCR product and the 2668 bp cleaved vectorwere separated by agarose gel electrophoresis and the corresponding DNAfragments were excised. The DNA was purified by means of the Qiagen GelPurification Kit following the manufacturer's instructions. Thereafter,vector and PCR product were ligated. The Rapid Ligation Kit from Rochewas used for this purpose. The resulting plasmid pUC19-Cnl1-C wasverified by sequencing.

In the next step, the OCS terminator (Genbank Accession V00088; DeGreve, H., et al. (1982) J. Mol. Appl. Genet. 1 (6): 499-511) wasamplified from the vector pGPVT-USP/OCS (DE 102 05 607) using thefollowing primers:

OCS_C 5′: (SEQ ID NO: 202) aggcctccatggcctgctttaatgagatatgcgagacgccOCS_C 3′: (SEQ ID NO: 203) cccgggccggacaatcagtaaattgaacggag

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme StuI and then for 12 hours at 25° C. with therestriction enzyme SmaI. The vector pUC19-Cnl1-C was incubated for 12hours at 25° C. with the restriction enzyme SmaI. Thereafter, the PCRproduct and the cleaved vector were separated by agarose gelelectrophoresis and the corresponding DNA fragments were excised. TheDNA was purified by means of the Qiagen Gel Purification Kit followingthe manufacturer's instructions. Thereafter, vector and PCR product wereligated. The Rapid Ligation Kit from Roche was used for this purpose.The resulting plasmid pUC19-Cnl1-C_OCS was verified by sequencing.

In the next step, the Cnl1-B promoter was amplified by PCR by means ofthe following primers:

Cnl1-B 5′: (SEQ ID NO: 204) aggcctcaacggttccggcggtatag Cnl1-B 3′:(SEQ ID NO: 205) cccggggttaacgctagcgggcccgatatcggatcccattttttggtggtgattggttct

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme StuI and then for 12 hours at 25° C. with therestriction enzyme SmaI. The vector pUC19-Cnl1-C was incubated for 12hours at 25° C. with the restriction enzyme SmaI. Thereafter, the PCRproduct and the cleaved vector were separated by agarose gelelectrophoresis and the corresponding DNA fragments were excised. TheDNA was purified by means of the Qiagen Gel Purification

Kit following the manufacturer's instructions. Thereafter, vector andPCR product were ligated. The Rapid Ligation Kit from Roche was used forthis purpose. The resulting plasmid pUC19-Cnl1-C_Cnl1B_OCS was verifiedby sequencing.

In a further step, the OCS terminator for Cnl1B was inserted. To thisend, the PCR was carried out using the following primers:

OCS2 5′: (SEQ ID NO: 206) aggcctcctgctttaatgagatatgcgagac OCS2 3′:(SEQ ID NO: 207) cccgggcggacaatcagtaaattgaacggag

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme StuI and then for 12 hours at 25° C. with therestriction enzyme SmaI. The vector pUC19-Cnl1C_Cnl1B_OCS was incubatedfor 12 hours at 25° C. with the restriction enzyme SmaI. Thereafter, thePCR product and cleaved vector were separated by agarose gelelectrophoresis and the corresponding DNA fragments were excised. TheDNA was purified by means of the Qiagen Gel Purification Kit followingthe manufacturer's instructions. Thereafter, vector and PCR product wereligated. The Rapid Ligation Kit from Roche was used for this purpose.The resulting plasmid pUC19-Cnl1-C_Cnl1B_OCS2 was verified bysequencing.

In the next step, the Cnl1-A promoter is amplified by PCR using thefollowing primers:

Cnl1-B 5′: (SEQ ID NO: 208) aggcctcaacggttccggcggtatagag Cnl1-B 3′:(SEQ ID NO: 209) aggccttctagactgcaggcggccgcccgcattttttggtggtgattggt

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was incubated for 2 hours at 37° C. with the restrictionenzyme StuI. The vector pUC19-Cnl1-C was incubated for 12 hours at 25°C. with the restriction enzyme SmaI. Thereafter, the PCR product andcleaved vector were separated by agarose gel electrophoresis and thecorresponding DNA fragments were excised. The DNA was purified by meansof the Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and PCR product were ligated. The RapidLigation Kit from Roche was used for this purpose. The resulting plasmidpUC19-Cnl1C_Cnl1B_Cnl1 A_OCS2 was verified by sequencing.

In a further step, the OCS terminator for Cnl1A was inserted. To thisend, the PCR was carried out with the following primers:

OCS2 5′: (SEQ ID NO: 210) ggcctcctgctttaatgagatatgcga OCS2 3′:(SEQ ID NO: 211) aagcttggcgcgccgagctcgtcgacggacaatcagtaaattgaacggaga

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme StuI and then for 2 hours at 37° C. with therestriction enzyme HindIII. The vector pUC19-Cnl1C_Cnl1B_Cnl1A_OCS2 wasincubated for 2 hours at 37° C. with the restriction enzyme StuI and for2 hours at 37° C. with the restriction enzyme HindIII. Thereafter, thePCR product and cleaved vector were separated by agarose gelelectrophoresis and the corresponding DNA fragments were excised. TheDNA was purified by means of the Qiagen Gel Purification Kit followingthe manufacturer's instructions. Thereafter, vector and PCR product wereligated. The Rapid Ligation Kit from Roche was used for this purpose.The resulting plasmid pUC19-Cnl1-C_Cnl1B_Cnl1A_OCS3 was verified bysequencing.

In the next step, the plasmid pUC19-Cnl1C_Cnl1B_Cnl1A_OCS3 was used forcloning the Δ6-, Δ5-desaturase and Δ6-elongase. To this end, the Phytiumirregulare Δ6-desaturase (WO02/26946) was amplified using the followingPCR primers:

D6Des(Pir) 5′: (SEQ ID NO: 212) agatctatggtggacctcaagcctggagtgD6Des(Pir) 3′: (SEQ ID NO: 213) ccatggcccgggttacatcgctgggaactcggtgat

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme BglII and then for 2 hours at 37° C. with therestriction enzyme NcoI. The vector pUC19-Cnl1C_Cnl1B_Cnl1A_OCS3 wasincubated for 2 hours at 37° C. with the restriction enzyme BglII andfor 2 hours at 37° C. with the restriction enzyme NcoI. Thereafter, thePCR product and cleaved vector were separated by agarose gelelectrophoresis and the corresponding DNA fragments were excised. TheDNA was purified by means of the Qiagen Gel Purification Kit followingthe manufacturer's instructions. Thereafter, vector and PCR product wereligated. The Rapid Ligation Kit from Roche was used for this purpose.The resulting plasmid pUC19-Cnl1_d6Des(Pir) was verified by sequencing.

In the next step, the plasmid pUC19-Cnl1_d6Des(Pir) was used for cloningthe Thraustochytrium ssp. Δ5-desaturase (WO02/26946). To this end, theThraustochytrium ssp. Δ5-desaturase was amplified using the followingPCR primers:

D5Des(Tc) 5′: (SEQ ID NO: 214) gggatccatgggcaagggcagcgagggccgD5Des(Tc) 3′: (SEQ ID NO: 215) ggcgccgacaccaagaagcaggactgagatatc

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme BamHI and then for 2 hours at 37° C. with therestriction enzyme EcoRV. The vector pUC19-Cnl1_d6Des(Pir) was incubatedfor 2 hours at 37° C. with the restriction enzyme BamHI and for 2 hoursat 37° C. with the restriction enzyme EcoRV. Thereafter, the PCR productand cleaved vector were separated by agarose gel electrophoresis and thecorresponding DNA fragments were excised. The DNA was purified by meansof the Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and PCR product were ligated. The RapidLigation Kit from Roche was used for this purpose. The resulting plasmidpUC19-Cnl1_d6Des(Pir)_d5Des(Tc) was verified by sequencing.

In the next step, the plasmid pUC19-Cnl1_d6Des(Pir)_d5Des(Tc) was usedfor cloning the Physcomitrella patens Δ6-elongase (WO01/59128), forwhich purpose the latter was amplified using the following PCR primers:

D6Elo(Pp) 5′: (SEQ ID NO: 216) gcggccgcatggaggtcgtggagagattctacggtgD6Elo(Pp) 3′: (SEQ ID NO: 217) gcaaaagggagctaaaactgagtgatctaga

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was first incubated for 2 hours at 37° C. with therestriction enzyme NotI and then for 2 hours at 37° C. with therestriction enzyme XbaI. The vector pUC19-Cnl1_d6Des(Pir)_d5Des(Tc) wasincubated for 2 hours at 37° C. with the restriction enzyme NotI and for2 hours at 37° C. with the restriction enzyme XbaI. Thereafter, the PCRproduct and cleaved vector were separated by agarose gel electrophoresisand the corresponding DNA fragments were excised. The DNA was purifiedby means of the Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and PCR product were ligated. The RapidLigation Kit from Roche was used for this purpose. The resulting plasmidpUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp) was verified by sequencing.

The binary vector for the transformation of plants was prepared startingfrom pUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp). To this end,pUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp) was incubated for 2 hours at37° C. with the restriction enzyme AscI. The vector pGPTV was treated inthe same manner. Thereafter, the fragment frompUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp) and the cleaved pGPTV vectorwere separated by agarose gel electrophoresis and the corresponding DNAfragments were excised. The DNA was purified by means of Qiagen GelPurification Kit following the manufacturer's instructions. Thereafter,vector and PCR product were ligated. The Rapid Ligation Kit from Rochewas used for this purpose. The resulting plasmidpGPTV-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp) was verified by sequencing.

A further construct,pGPTV-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co), was used. To thisend, the amplification was carried out with the following primers,starting from pUC19-Cnl1C_OCS:

Cnl1_OCS 5′: (SEQ ID NO: 218) gtcgatcaacggttccggcggtatagagttgCnl1_OCS 3′: (SEQ ID NO: 219) gtcgatcggacaatcagtaaattgaacggaga

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl2

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was incubated for 2 hours at 37° C. with the restrictionenzyme SalI. The vector pUC19 was incubated for 2 hours at 37° C. withthe restriction enzyme SalI. Thereafter, the PCR product and the cleavedvector were separated by agarose gel electrophoresis and thecorresponding DNA fragments were excised. The DNA was purified by meansof Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and PCR product were ligated. The RapidLigation Kit from Roche was used for this purpose. The resulting plasmidpUC19-Cnl1 OCS was verified by sequencing.

In a further step, the Calendula officinalis Δ12-desaturase gene(WO01/85968) was cloned into pUC19-Cnl1_OCS. To this end, d12Des(Co) wasamplified with the following primers:

D12Des(Co) 5′: (SEQ ID NO: 220) agatctatgggtgcaggcggtcgaatgcD12Des(Co) 3′: (SEQ ID NO: 221) ccatggttaaatcttattacgatacc

Composition of the PCR mix (50 μl):

5.00 μl template cDNA

5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl₂

5.00 μl of 2 mM dNTP

1.25 μl of each primer (10 pmol/μl)

0.50 μl of Advantage polymerase (Clontech)

PCR reaction conditions:

Annealing temperature: 1 min 55° C.

Denaturation temperature: 1 min 94° C.

Elongation temperature: 2 min 72° C.

Number of cycles: 35

The PCR product was incubated for 2 hours at 37° C. with the restrictionenzyme BglII and thereafter for 2 hours at the same temperature withNcoI. The vector pUC19-Cnl1 OCS was incubated in the same manner.Thereafter, the PCR fragment and the cleaved vector were separated byagarose gel electrophoresis and the corresponding DNA fragments wereexcised. The DNA was purified by means of Qiagen Gel Purification Kitfollowing the manufacturer's instructions. Thereafter, vector and PCRproduct were ligated. The Rapid Ligation Kit from Roche was used forthis purpose. The resulting plasmid pUC19-Cnl1 D12Des(Co) was verifiedby sequencing.

The plasmid pUC19-Cnl1 D12Des(Co) and the plasmidpUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp) were incubated for 2 hours at37° C. with the restriction enzyme SalI. Thereafter, the vector fragmentand the cleaved vector were separated by agarose gel electrophoresis andthe corresponding DNA fragments were excised. The DNA was purified bymeans of Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and vector fragment were ligated. TheRapid Ligation Kit from Roche was used for this purpose. The resultingplasmid pUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) wasverified by sequencing.

The binary vector for the transformation of plants was prepared startingfrom pUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co). To this end,pUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) was incubated for 2hours at 37° C. with the restriction enzyme AscI. The vector pGPTV wastreated in the same manner. Thereafter, the fragment frompUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) and the cleavedpGPTV vector were separated by agarose gel electrophoresis and thecorresponding DNA fragments were excised. The DNA was purified by meansof Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, vector and PCR product were ligated. The RapidLigation Kit from Roche was used for this purpose. The resulting plasmidpGPTV-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) was verified bysequencing.

A further example of the use of seed-specific expression constructs isthe Napin promoter. Preparation of these expression constructs in thevectors pGPTV or pSUN is described in Wu et al. (2005) Nat. Biotech.23:1013-1017.

A further vector suitable for plant transformation is pSUN2. This vectorwas used in combination with the Gateway system (Invitrogen, Karlsruhe)in order to increase the number of expression cassettes present in thevector to more than four. For this purpose, the Gateway cassette A wasinserted into the vector pSUN2 in accordance with the manufacturer'sinstructions, as described below:

The pSUN2 vector (1 μg) was incubated with the restriction enzyme EcoRVat 37° for 1 h. The Gateway cassette A (Invitrogen, Karlsruhe) was thenligated into the cut vector using the Rapid Ligation kit from Roche,Mannheim. The resulting plasmid was transformed into E. coli DB3.1 cells(Invitrogen). The isolated plasmid pSUN-GW was then verified bysequencing.

In the second step, the expression cassette was cut out ofpUC19-Cnl1_d6Des(Pir)_d5Des(Tc)_D6Elo(Pp)_D12Des(Co) using AscI andligated into the likewise treated vector pSUN-GW. The plasmid obtainedin this way pSUN-4G was used for further gene constructs.

For this purpose, firstly a pENTR clone was modified in accordance withthe manufacturer's instructions (Invitrogen). The plasmid pENTR1A(Invitrogen) was incubated with the restriction enzyme EcoRI at 37° for1 h and then treated with Klenow enzyme and with a 1 μM dNTP mix for 30min, and subsequently the AscI adapter (5′-ggcgcgcc; phosphorylated atthe 5′ end, double-stranded) was ligated into the pENTR1A vector. Geneswere inserted as described above stepwise into the Cnl cassette in thesemodified and transferred via AscI into the pENTR vector, resulting inthe pENTR-Cn1 vector.

In a further step, the pSUN-8G construct was prepared. For this purpose,5′ and 3′ primers for the genes with the SEQ ID NOs: 1, 3, 5 and 7 withthe restriction cleavage sites described above and with the first and ineach case last 20 nucleotides of the open reading frame were producedand amplified with the standard conditions (see above) and ligated intothe pENTR-Cnl vector, which was subsequently subjected to arecombination reaction with the pSUN-4G vector in accordance with themanufacturer's instructions.

The construct pSUN-8G was prepared in this way and was transformed intoBrassica juncea and Brassica napus. The seeds of the transgenic plantswere analyzed by gas chromatography.

A further construct which was used for transformation of B. juncea andB. napus was the construct pSUN-9G. This construct was preparedaccording to Wu et al. (2005) Nat. Biotech. 23:1013-1017 with the napinpromoter. In a modification of Wu et al. 2005, the coding sequence ofOtELO2.2 was inserted in the described manner instead of the gene OmELO.The resulting construct pSUN-9G was then transformed into B. juncea andB. napus.

Example 8: Lipid Extraction from Plant Material

The effect of the genetic modification in plants on the production of adesired compound (such as a fatty acid) can be determined by growing themodified plant under suitable conditions (such as those described above)and analyzing the medium and/or the cellular components for the elevatedproduction of the desired product (i.e. of the lipids or a fatty acid).These analytical techniques are known to the skilled worker and comprisespectroscopy, thin-layer chromatography, various types of stainingmethods, enzymatic and microbiological methods and analyticalchromatography such as high-performance liquid chromatography (see, forexample, Ullman, Encyclopedia of Industrial Chemistry, Vol. Δ2, p. 89-90and p. 443-613, VCH: Weinheim (1985); Fallon A. et al. (1987)“Applications of HPLC in Biochemistry” in: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)Biotechnology, Vol. 3, Chapter III: “Product recovery and purification”,p. 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:downstream processing for Biotechnology, John Wiley and Sons; Kennedy,J. F., and Cabral, J. M. S. (1992) Recovery processes for biologicalMaterials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D.(1988) Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned methods, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940 and Browse et al. (1986) AnalyticBiochemistry 152:141-145. The qualitative and quantitative analysis oflipids or fatty acids is described by Christie, William W., Advances inLipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library;2); Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for the production of the desired compound, such as intermediatesand by-products, in order to determine the overall production efficiencyof the compound. The analytical methods comprise measuring the amount ofnutrients in the medium (for example sugars, hydrocarbons, nitrogensources, phosphate and other ions), measuring the biomass compositionand the growth, analyzing the production of conventional metabolytes ofbiosynthetic pathways and measuring gases which are generated during thefermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, Ed., IRL Press, p. 103-129; 131-163 and 165-192 (ISBN:0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

The unambiguous detection for the presence of fatty acid products can beobtained by analyzing recombinant organisms using analytical standardmethods: GC, GC-MS or TLC, as described on several occasions by Christieand the references therein (1997, in: Advances on Lipid Methodology,Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,Gaschromatographie-Massenspektrometrie-Verfahren [Gaschromatography/mass spectrometric methods], Lipide 33:343-353).

The material to be analyzed can be disrupted by sonication, grinding ina glass mill, liquid nitrogen and grinding or via other applicablemethods. After disruption, the material must be centrifuged. Thesediment is resuspended in distilled water, heated for 10 minutes at100° C., cooled on ice and recentrifuged, followed by extraction for onehour at 90° C. in 0.5 M sulfuric acid in methanol with 2%dimethoxypropane, which leads to hydrolyzed oil and lipid compounds,which give transmethylated lipids. These fatty acid methyl esters areextracted in petroleum ether and finally subjected to a GC analysisusing a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25m, 0.32 mm) at a temperature gradient of between 170° C. and 240° C. for20 minutes and 5 minutes at 240° C. The identity of the resulting fattyacid methyl esters must be defined using standards which are availablefrom commercial sources (i.e. Sigma).

Plant material is initially homogenized mechanically by comminuting in apestle and mortar to make it more amenable to extraction.

This is followed by heating at 100° C. for 10 minutes and, after coolingon ice, by resedimentation. The cell sediment is hydrolyzed for one hourat 90° C. with 1 M methanolic sulfuric acid and 2% dimethoxypropane, andthe lipids are transmethylated. The resulting fatty acid methyl esters(FAMEs) are extracted in petroleum ether. The extracted FAMEs areanalyzed by gas liquid chromatography using a capillary column(Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and atemperature gradient of from 170° C. to 240° C. in 20 minutes and 5minutes at 240° C. The identity of the fatty acid methyl esters isconfirmed by comparison with corresponding FAME standards (Sigma). Theidentity and position of the double bond can be analyzed further bysuitable chemical derivatization of the FAME mixtures, for example togive 4,4-dimethoxyazolin derivatives (Christie, 1998) by means of GC-MS.

Example 9: Use of the Optimized Δ5-Elongase (as Depicted in SEQ ID NO:64) from Ostreococcus tauri for Constructs for Constitutive Expression

Transformation vectors based on pGPTV-35S, a plasmid based on pBIN19-35S(Bevan M. (1984) Nucl. Acids Res. 18:203), were produced for thetransformation of plants. For this purpose, firstly an expressioncassette consisting of the promoter element CaMV35S (SEQ ID NO: 161) andthe 35S terminator (SEQ ID NO: 162; Franck, A. et al. (1980) Cell 21(1): 285-294) was assembled in a pUC vector. This entailed the promoterbeing inserted via the SalI/XbaI restriction cleavage sites and theterminator via the BamHI/SmaI restriction cleavage sites. In addition, apolylinker with the XhoI cleavage site was attached to the terminator(‘triple ligation’). The resulting plasmid pUC19-35S was then employedfor cloning PUFA genes. In parallel, the open reading frames of theΔ6-desaturase (SEQ ID NO: 1), of the Δ5-desaturase (SEQ ID NO: 51) andΔ6-elongase (SEQ ID NO: 171) sequences were inserted via the EcoRVcleavage site into pUC19-35S vectors. The resulting plasmids pUC-D6,pUC-D5, pUC-E6(Tc) were used to construct the binary vector pGPTV-35SD6D5E6(Tc). For this purpose, the vector pGPTV was digested with theenzyme SalI, the plasmid pUC-D6 was digested with SalI/XhoI, and thecorrect fragments were ligated. The resulting plasmid pGPTV-D6 was thendigested with SalI, the plasmid pUC-D5 was digested with SalI/XhoI, andthe correct fragments were ligated. The resulting plasmid pGPTV-D6-D5was then digested once more with SalI, the plasmid pUC-E6(Tc) withSalI/XhoI, and the correct fragments were ligated. These sequentialcloning steps resulted in the binary vector pGPTV-D6D5E6(Tc), which wasemployed for the transformation.

In a further procedure, the sequence of d6Elo(Tp) (SEQ ID NO: 163) wasinserted into the vector pUC19-35S instead of the sequence d6Elo(Tc).The resulting plasmid pUC-E6(Tp) was used to prepare the binary vectorpGPTV-35S D6D5E6(Tp).

In a further procedure, the open reading frame of ω3Des (SEQ ID NO: 193)was cloned into pUC19-35S. The resulting plasmid pUC-ω3Pi wastransferred via SalI/XhoI into the binary vectors pGPTV-D6D5E6(Tc) andpGPTV-D6D5E6(Tp). The resulting vectors pGPTV-D6D5E6(Tc)ω3Pi andpGPTV-D6D5E6(Tp)ω3Pi were employed for the plant transformation.

In a further procedure, the open reading frame of the optimizedΔ5-elongase from Ostreococcus tauri (SEQ ID NO: 64) and the open readingframe of the Δ4-desaturase from Thraustochytrium sp. (SEQ ID NO: 77) wascloned into pUC19-35S. The resulting plasmids pUC-E5 and pUC-D4 werethen transferred via SalI/XhoI in accordance with the above statementsinto the vector pGPTV-D6D5E6(Tp)ω3Pi. The resulting vectorpGPTV-D6D5E6(Tp)ω3PiE5D4 was employed for the plant transformation.

All the binary vectors were transformed into E. coli DH5a cells(Invitrogen) in accordance with the manufacturer's instructions.Positive clones were identified by PCR, and plasmid DNA was isolated(Qiagen Dneasy).

Example 10: Transformation of the Constitutive Binary Vectors intoPlants

-   a) Generation of transgenic Brassica napus and Brassica juncea    plants. The protocol for the transformation of oilseed rape plant    was used (modification of Moloney et al. (1992) Plant Cell Reports    8:238-242)

The binary vector pGPTV-D6D5E6(Tp)ω3PiE5D4 was transformed inAgrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al. (1984) Nucl.Acids. Res. 13: 4777-4788). A 1:50 dilution of an overnight culture of apositively transformed agrobacterial colony in Murashige-Skoog medium(Murashige and Skoog (1962) Physiol. Plant. 15: 473) supplemented with3% sucrose (3MS medium) was used for the transformation ofOrychophragmus violaceus. Petioles or hypocotyls of freshly germinatedsterile plants (in each case approx. 1 cm²) were incubated with a 1:50agrobacterial dilution for 5-10 minutes in a Petri dish. This isfollowed by 3 days of coincubation in the dark at 25° C. on 3MS mediumsupplemented with 0.8% Bacto agar. Thereafter, the cultivation wascontinued with 16 hours light/8 hours dark and a weekly rhythm on MSmedium supplemented with 500 mg/l Claforan (cefotaxime-sodium), 50 mg/lkanamycin, 20 μM benzylaminopurine (BAP) and 1.6 g/l glucose. Growingshoots were transferred to MS medium supplemented with 2% sucrose, 250mg/l Claforan and 0.8% Bacto agar. If no roots had developed after threeweeks, 2-indolebutyric acid was added to the medium as growth hormonefor rooting.

Regenerated shoots were obtained on 2MS medium with kanamycin andClaforan, then, after rooting, transferred into soil and, aftercultivation, grown for two weeks in a controlled-environment cabinet orin the greenhouse, allowed to flower, mature seeds were harvested andanalyzed for elongase expression such as Δ6-elongase activity or for Δ5-or Δ6-desaturase activity by means of lipid analyses. In this manner,lines with elevated contents of polyunsaturated C20- and C22-fatty acidswere identified.

-   b) Generation of transgenic Orychophragmus violaceus plants

The protocol for the transformation of oilseed rape plants was used(modification of Moloney et al. (1992) Plant Cell Reports 8:238-242) asdescribed under a).

To generate transgenic plants, the binary vectorpGPTV-D6D5E6(Tp)ω3PiE5D4 was transformed into Agrobacterium tumefaciensC58C1:pGV2260 (Deblaere et al. (1984) Nucl. Acids. Res. 13: 4777-4788).A 1:50 dilution of an overnight culture of a positively transformedAgrobacterium colony in Murashige-Skoog medium (Murashige and Skoog(1962) Physiol. Plant, 15: 473) with 3% sucrose (3MS medium) was used totransform Orychophragmus violaceus. Petioles or hypocotyls of freshlygerminated sterile plants (each about 1 cm²) were incubated with a 1:50agrobacterial dilution in a Petri dish for 5-10 minutes. This isfollowed by coincubation on 3MS medium with 0.8% Bacto agar in the darkat 25° C. for 3 days. The cultivation was then continued with 16 hourslight/8 hours dark and in a weekly rhythm on MS medium with 500 mg/lClaforan (cefotaxime sodium), 15 mg/l kanamycin, 20 μM benzylaminopurine(BAP) and 1.6 g/l glucose. Growing shoots were transferred to MS mediumwith 2% sucrose, 250 mg/l Claforan and 0.8% Bacto agar. If no roots haddeveloped after three weeks, 2-indolebutyric acid was added to themedium as growth hormone for rooting.

Regenerated shoots were obtained on 2MS medium with kanamycin andClaforan and, after rooting, transferred to soil and, after cultivation,grown for two weeks in a controlled environment cabinet or in agreenhouse, allowed to flower, and mature seeds were harvested andexamined by lipid analyses for elongase expression such as Δ6-elongaseactivity or Δ5- or Δ6-desaturase activity. Lines with increased contentsof polyunsaturated C20 and C22 fatty acids were identified in this way.

-   c) Transformation of Arabidopsis thaliana plants

The protocol of Bechthold et al. (1993) C. R. Acad. Sci. Ser. III Sci.Vie. 316: 1194-1199 was used.

To generate transgenic plants, the generated binary vectorpGPTV-D6D5E6(Tp)ω3PiE5D4 was transformed into Agrobacterium tumefaciensC58C1:pMP90 (Deblaere et al. (1984) Nucl. Acids. Res. 13: 4777-4788)and, in accordance with the protocol of Bechthold et al. (1993), flowersof Arabidopsis thaliana cv. Columbia 0 were dipped in an agrobacterialsolution with OD600=1.0. The procedure was repeated again two dayslater. Seeds from these flowers were then placed on agar plates with 1/2MS, 2% sucrose and 50 mg/l kanamycin. Green seedlings were thentransferred to soil.

Example 11: Analysis of Plant Material of Transgenic Orychophragmus orArabidopsis Plants

Extraction of leaf material of transgenic Orychophragmus violaceus andArabidopsis thaliana plants transformed with pGPTV-D6D5E6(Tp)ω3PiE5D4and the gas chromatography analysis was carried out as described inexample 8. Table 2 shows the results of the analyses. The various fattyacids are indicated in percent by weight. It was possible to show thatlong-chain polyunsaturated fatty acids were synthesized by bothdifferent plant species. It was surprisingly possible with the optimizedsequence of the Δ5-elongase (as depicted in SEQ ID NO: 64) fromOstreococcus tauri to obtain a distinctly higher yield of DHA thanreported for example by Robert et al. (2005) Functional Plant Biology32: 473-479 for Arabidopsis thaliana with 1.5% DHA. It was possible forthe first time to achieve a synthesis of long-chain polyunsaturatedfatty acids for Orychophragmus violaceus.

Example 12: Analysis of Seeds of Transgenic Brassica juncea Lines

Extraction of seeds of transgenic Brassica juncea plants transformedwith pSUN-9G, and the gas chromatography analysis was carried out asdescribed in example 8. Table 6 shows the results of the analyses. Thevarious fatty acids are indicated in percent area. As in Wu et al. 2005it was possible to show the synthesis of long-chain polyunsaturatedfatty acids (PUFA). Surprisingly, the use of the modified elongasesequence OtELO2.2 such as the nucleic acid sequence described by SEQ IDNO: 64 resulted in a drastic increase in the content of C22 fatty acids.In total, the seed oil contained about 8% by weight % polyunsaturatedC22 fatty acids. Specifically, the content of the fatty aciddocosahexaenoic acid (DHA) in the seed oil was 1.9% by weight %,representing an increase by a factor of 10 compared with Wu et al. 2005.

Example 13: Detailed Analysis of the Lipid Classes and Position Analysisof Leaf Material from O. violaceus

About 1 g of leaf tissue was heated in 4 ml of isopropanol at 95° C. for10 minutes, homogenized by Polytron and shaken after addition of 1.5 mlof chloroform. The samples were centrifuged, the supernatant wascollected, and the pellet was extracted again withisopropanol:chloroform 1:1 (v/v). The two extracts were combined, driedand dissolved in chloroform. The lipid extract was prefractionated on asilica prepsep column (Fisher Scientific, Nepean, Canada) into neutrallipids, glycolipids and phospholipids, eluting with chloroform:aceticacid 100:1 (v/v), acetone:acetic acid 100:1 (v/v) andmethanol:chloroform:water 100:50:40 (v/v/v), respectively. Thesefractions were further fractionated on silica G-25 thin-layerchromatography plates (TLC; Macherey-Nagel, Duren, Germany). Neutrallipids were developed with hexane:diethyl ether:acetic acid (70:30:1),glycolipids with chloroform:methanol:ammonia (65:25:4 v/v/v) andphospholipids with chloroform:methanol:ammonia:water (70:30:4:1v/v/v/v). The individual lipid classes were identified after sprayingwith primulin under UV light, removed by scraping off the plates andeither used for direct transmethylation or extracted by a suitablesolvent for further analysis.

It was possible by the disclosed methods for the various lipid classes(neutral lipids, phospho-lipids and galactolipids) to be fractionatedand analyzed separately. The glycolipids were additionally examined forthe position of the individual fatty acids.

a) Regiospecific Analysis of the Triacylglycerides (TAG)

Three to five mg of the TLC-purified TAG were dried under nitrogen in aglass tube, resuspended in aqueous buffer by brief ultrasound treatment(1 M Tris pH 8; 2.2% CaCl₂) (w/v); 0.05% bile salts (w/v)) and incubatedat 40° C. for 4 minutes. After addition of 0.1 ml of a solution ofpancreatic lipase (10 mg/ml in water), the samples were vigorouslyvortexed for 3 minutes, and the digestion was stopped by adding 1 ml ofethanol and 1.5 ml of 4 M HCl. The partly digested TAGs were extractedtwice with diethyl ether, washed with water, dried and dissolved in asmall volume of chloroform. Monoacylglycerols (MAG) were separated fromthe free fatty acids and undigested TAGs on a TLC plate as describedabove for neutral lipids. The point corresponding to the MAGs wasanalyzed by GC and represented the sn-2 position of the TAGs. Thedistribution of the fatty acids to the remaining sn-1 and sn-3 positionswas calculated by the following formula: sn-1+sn-3=(TAG×3−MAG)/2.

This position analysis of the triacylglycerides revealed in this casethat EPA and DHA are present in similar concentrations in the sn-2 andsn-1/3 positions, while ARA is to be found overall only in small amountsin the triacylglycerides, and here mainly in the sn-2 position (Tab. 3).

b) Stereospecific Analysis of Phospholipids

Fractionated and extracted phosphatidylglycol (PG),phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were driedunder N₂ and resuspended in 0.5 ml of borate buffer (0.5M, pH 7.5,containing 0.4 mM CaCl₂). After a brief ultrasound treatment, 5U ofphospholipase Δ2 from the venom of Naja mossambica (Sigma P-7778) and 2ml of diethyl ether were added and the samples were vortexed at roomtemperature for 2 hours. The ether phase was dried, the digestion wasstopped with 0.3 ml of 1M HCl, and the reaction mixture was extractedwith chloroform:methanol (2:1 v/v). The digested phospholipids wereseparated by TLC in chloroform:methanol:ammonia:water (70:30:4:2v/v/v/v) and points which corresponded to the liberated free fatty acidsand lysophospholipids were removed by scraping and directlytransmethylated.

Positional analysis of the phospholipids showed an accumulation of EPAand DHA in the sn-2 position of phosphatidylcholine (PC), while DHA wassimilarly distributed in sn-1 and sn-2 position inphosphatidylethanolamine (PE). Only traces of, or no, ARA was to befound in both phospholipids (Tab. 4). The concentrations of EPA and DHAin phosphatidylglycerol were lower than in the other investigatedphospholipids, with accumulation in the sn-2 position also to beobserved in this lipid class (Tab. 4, PG).

c) Stereospecific Analysis of Glycolipids

The galactolipids were investigated as a further polar lipid class.Galactolipids are found in the membranes of plastids and form the maincomponents there.

TLC-purified monogalactosyldiacylglycerol (MGDG) anddigalactosyldiacylglycerol (DGDG) were dried under nitrogen anddissolved in 0.5 ml of diethyl ether. Then 25 units of the lipase fromRhizopus arrhizus (Sigma 62305), resuspended in 2 ml of borate buffer(50 mM, pH 7.5 containing 2 mM CaCl₂), were added, and the samples werevortexed at room temperature for 2 hours. The ether phase was dried andthe digestion was stopped by adding 0.3 ml of 1M HCl, and the lipidswere extracted with 4 ml of chloroform:methanol (2:1 v/v). After drying,the digested galactolipids were in a small volume of chloroform:methanol(2:1 v/v) and developed twice on a precoated silica TLC plate, firstlywith chloroform:methanol: ammonia:water (70:30:4:1 v/v/v/v) to about twothirds the height of the plate, followed by complete development inhexane:diethyl ether:acetic acid (70:30:1). The points whichcorresponded to the liberated free fatty acids and the lysogalactolipidswere identified after spraying with primulin, scraped off andtransmethylated directly for GC analysis.

It was possible to find VLCPUFA (very long chain polyunsaturated fattyacid) in these lipids too, with an accumulation of EPA in the sn-2position being observed. DHA was to be found only in thedigalactodiacylglycerols (DGDG) and was undetectable in themonogalactodiacylglycerols (MGDG) (Table 5). The distribution of VLCPUFAin galactolipids, a compartment in which these fatty acids were notexpected, shows the dynamics of the synthesis and the latertransformation. VLCPUFA in polar lipids are of particular nutritionalvalue because they can be absorbed better in the intestines of mammalsthan the neutral lipids.

TABLE 1 Test of the optimized sequences of pOTE1.1 and pOTE2.1 in yeast.The conversion rates were determined in accordance with the substrateconversions. A distinct rise in activity was achievable with theoptimized sequence in plasmid pOTE2.2. Conversion rates of theOstreococcus tauri elongases Substrate GLA ARA EPA Genes Product 20:322:4 22:5 pOTE1.1 d6Elongase(Ot) 21.1 pOTE1.2 d6Elongase(Ot)_opt 25.6pOTE2.1 d5Elongase(Ot) 7.3 35.9 pOTE2.2 d5Elongase(Ot)_opt 32.7 63.1

TABLE 2 Gas chromatographic analysis of leaf material of Orychophragmusviolaceus and Arabidopsis thaliana. The individual fatty acids areindicated in percent area. Fatty acids 16:0 16:3 18:1 18:2 GLA 18:3 18:4ARA EPA DPA DHA Fatty acid composition of leaf material ofOrychophragmus violaceus Control 20.9 8.5 3.3 16.0 0.0 47.4 0.0 0.0 0.00.0 0.0 Trans- 21.3 8.2 5.2 5.2 4.2 23.1 5.0 0.6 13.5 2.7 4.5 gene Fattyacid composition of leaf material of Arabidopsis thaliana Control 12.810.0 3.5 14.2 0.0 54.6 0.0 0.0 0.0 0.0 0.0 Trans- 19.3 8.5 5.0 4.6 6.431.0 4.4 0.0 6.3 1.5 6.3 gene

TABLE 3 Regiospecific analysis of the triacylglycerides from leafmaterial from transgenic O. violaceus plants. TAG 16:0 18:0 18:1n−918:2n−9 18:2n−6 18:3n−6 18:3n−3 18:4n−3 20:3n−6 20:4n−6 20:4n−3 20:5n−322:5n−3 22:6n−3 wt 25.12 3.03 5.06 18.53 44.72 sn−2 1.42 0.76 6.79 27.6262.03 sn−1 + 3 36.97 4.17 4.19 13.98 36.07 Trans- 22.63 3.12 3.45 0.772.35 9.51 6.37 13.03 0.74 0.83 3.87 24.96 2.22 4.15 gene sn−2 1.62 0.648.33 1.61 5.15 16.21 10.88 19.84 0.17 1.38 1.99 24.82 3.27 3.02 sn−1 + 333.13 4.36 1.02 0.35 0.96 6.16 4.11 9.63 1.02 0.55 4.80 25.03 1.69 4.72

TABLE 4 Stereospecific analysis of the phospholipids from leaf materialfrom transgenic O. violaceus plants. 16:0 16:1 18:0 18:1n−9 18:1n−718:2n−9 18:2n−6 18:3n−6 18:3n−3 PG wt 27.96 20.04 4.11 2.89 0.90 21.820.00 21.56 sn−2 17.26 0.53 2.61 3.82 1.91 39.01 0.00 34.44 sn−1 38.6639.56 5.62 1.96 0.00 4.62 0.00 8.69 Trans- 27.15 24.70 3.08 4.62 1.200.00 15.15 1.53 17.94 gene sn−2 21.16 3.61 4.23 7.52 2.14 27.40 0.5031.57 sn−1 33.15 45.79 1.94 1.71 0.27 2.90 2.57 4.30 PE wt 37.49 0.006.62 4.35 1.37 19.28 29.95 sn−2 54.22 0.00 7.74 3.39 3.42 12.64 13.71sn−1 20.77 0.00 5.51 5.31 0.00 25.93 46.18 Trans- 31.78 0.81 5.84 3.082.20 0.85 5.57 11.25 11.34 gene sn−2 50.17 0.33 10.86 3.22 4.94 0.352.63 3.27 3.59 sn−1 13.40 1.29 0.83 2.95 0.00 1.35 8.50 19.23 19.10 PCwt 27.67 0.84 6.38 8.56 1.80 21.75 33.01 sn−2 48.05 0.44 8.65 5.05 3.4114.52 18.04 sn−1 7.28 1.24 4.11 12.06 0.18 28.97 47.98 Trans- 21.00 0.008.01 10.02 2.86 1.25 3.77 11.63 5.60 gene sn−2 45.35 0.00 14.71 5.085.70 0.31 3.23 3.09 4.58 sn−1 3.36 0.00 1.30 14.96 0.02 2.20 4.31 20.186.62 18:4n−3 20:3n−6 20:4n−6 20:4n−3 20:5n−3 22:5n−3 22:6n−3 PG wt sn−2sn−1 Trans- 1.40 0.00 0.00 0.45 2.18 0.10 0.58 gene sn−2 0.81 0.38 1.240.00 0.33 sn−1 2.00 0.51 3.13 0.27 0.83 PE wt sn−2 sn−1 Trans- 7.38 0.000.00 2.88 9.41 1.90 4.90 gene sn−2 2.31 0.56 4.42 6.18 0.38 4.19 sn−112.45 0.00 1.34 12.64 3.41 5.61 PC wt sn−2 sn−1 Trans- 12.11 0.50 0.004.34 11.16 3.76 3.70 gene sn−2 2.65 0.61 0.08 4.01 8.32 0.41 1.18 sn−121.56 0.38 0.00 4.66 13.99 7.12 6.22

TABLE 5 Stereospecific analysis of the galactolipids from leaf materialfrom transgenic O. violaceus plants. 16:0 16:1 16:2 16:3 18:0 18:1n−918:1n−7 18:2n−9 18:2n−6 18:3n−6 18:3n−3 18:4n−3 20:3n−6 20:4n−6 20:4n−320:5n−3 MGDG wt 2.64 0.13 1.23 30.72 0.33 0.35 0.26 3.81 60.52 sn−2 0.000.05 0.00 7.11 0.35 0.31 0.41 4.60 87.30 sn−1 5.34 0.21 2.55 54.34 0.310.39 .012 3.01 33.74 Trans- 4.16 0.20 1.08 33.81 0.93 0.73 0.52 0.031.64 1.88 44.82 2.73 0.04 0.30 0.50 5.08 gene sn−2 1.22 0.29 0.54 4.791.51 1.15 0.93 0.00 2.80 0.14 80.19 0.00 0.08 0.17 0.87 3.86 sn−1 7.110.11 1.61 62.82 0.34 0.31 0.11 0.11 0.47 3.62 9.46 5.48 0.00 0.43 0.146.31 DGDG wt 17.67 0.19 0.38 2.15 1.61 0.51 0.94 5.56 70.71 sn−2 16.840.25 0.50 2.52 2.21 0.55 1.75 6.07 0.00 68.74 sn−1 18.50 0.12 0.27 1.781.01 0.46 0.13 5.05 72.68 Trans- 18.50 0.00 0.00 2.62 2.84 1.36 1.390.00 6.28 3.55 54.66 0.00 0.00 0.00 2.18 5.36 gene sn−2 22.74 0.17 0.230.48 4.55 1.71 2.32 0.24 9.22 0.23 56.06 0.27 0.00 0.00 0.36 1.23 sn−114.27 0.00 0.00 4.77 1.12 1.00 0.46 0.00 3.33 6.88 53.26 0.00 0.00 0.004.01 9.49

TABLE 6 Gas chromatographic determination of the fatty acids from seedsof transgenic Brassica juncea plants transformed with the constructpSUN-9G in percent by weight. WT describes the unmodified wild-typecontrol. Lipid Profile (%) 20:3 20:3 (8, 11, (11, 14, 16:0 18:0 18:118:2 γ18:3 α18:3 18:4 20:0 14) 17) BJ223_PUFA184_MKP71_581A 4.4 3.0 22.516.9 27.0 4.9 3.2 0.6 1.1 0.5 BJ223_PUFA184_MKP71_581A 4.7 3.9 17.9 10.629.5 4.2 4.0 0.9 2.0 0.9 BJ223_PUFA184_MKP71_581A 4.4 3.0 18.9 13.8 30.54.1 3.2 0.7 1.3 0.7 BJ223_PUFA184_MKP71_581A 4.6 3.3 20.5 13.2 29.8 4.23.3 0.8 1.4 0.6 Lipid Profile (%) 20:4 (ARA) 20:4 (ETeA) 20:5 (EPA) (5,8, (8, 11, (5, 8, 11, 14) 14, 17) 11, 14, 17) 22:1 22:4 22:5 22:6BJ223_PUFA184_MKP71_581A 3.1 0.6 4.6 0.0 1.5 2.0 1.5BJ223_PUFA184_MKP71_581A 4.2 1.0 4.1 0.0 3.1 3.5 1.9BJ223_PUFA184_MKP71_581A 4.1 0.5 4.5 0.0 2.7 2.8 1.6BJ223_PUFA184_MKP71_581A 3.6 0.6 4.4 0.0 2.4 2.5 1.6

1. A fatty acid mixture produced by a transgenic Brassica plant, saidfatty acid mixture comprising 30% to 80% by weight of esterifiedpolyunsaturated fatty acids, wherein said polyunsaturated fatty acidscomprise a total amount of 15% to 30% by weight of eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) based on the total fatty acids inthe fatty acid mixture in the form of triacylglycerides.
 2. A fatty acidmixture produced by a transgenic Brassica plant, said fatty acid mixturecomprising at least 60% by weight of esterified polyunsaturated fattyacids, wherein said polyunsaturated fatty acids comprise a total amountof 15% to 30% by weight of eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) based on the total fatty acids in the fattyacid mixture in the form of triacylglycerides.
 3. The fatty acid mixtureof claim 2, wherein the fatty acid mixture comprises at least 70% byweight of esterified polyunsaturated fatty acids.
 4. The fatty acidmixture of claim 2, wherein the fatty acid mixture comprises at least80% by weight of esterified polyunsaturated fatty acids.